Immunointeractive molecules and uses therefor

ABSTRACT

The present invention relates generally to immunointeractive molecules and their use inter alia in the detection and/or purification of T-cell antigen binding molecules (TABMs). The ability to determine the presence and levels of particular TABMs provides a useful diagnostic procedures for a variety of disease conditions.

FIELD OF THE INVENTION

[0001] The present invention relates generally to immunointeractivemolecules and their use inter alia in the detection and/or purificationof T-cell antigen binding molecules (TABM). These molecules are alsocalled T-cell derived antigen binding molecules. The ability todetermine the presence and levels of particular TABM provide a usefuldiagnostic monitoring procedure for a variety of disease or otherphysiological conditions which are associated directly or indirectlywith TABM including a range of allergies, immunological status,immunological dysfunction, neuropeptide release, infection, cancer,autoimmune disease or vaccination status. The immunointeractivemolecules of the present invention are also useful in a range ofconditions including modulating aspects of an immune response such asbut not limited to cell mediated immunity.

BACKGROUND OF THE INVENTION

[0002] Bibliographic details of the publications numerically referred toin this specification are collected at the end of the description.

[0003] The rapidly increasing knowledge of the immune system is greatlyfacilitating the rationale design of therapeutic and diagnosticprocedures based on modulating aspects and compounds of the immunesystem. Our understanding of the immune system is predicated in part onthe identification and characterization of immune system components anddetermining how various components interact. Although a substantialfocus of the research over the years has been on studyingimmunoglobulins, other immunointeractive molecules such as TABM havereceived less attention. This has been due, in part, to the inability topurify large amounts of these immunointeractive molecules.

[0004] TABM are antigen binding molecules generally located in theserum, derived from T-cells. TABM can be regarded as immunoproteinswhich are antigen specific and, hence, are analogous to immunoglobulins.TABM are, however, distinct from immunoglobulins. For example, TABMrecognise different epitopes to immunoglobulins. TABM are present inserum in multimeric form, the monomer having an apparent molecularweight on a 10-15% w/v SDS polyacrylamide gel of about 24,000-30,000daltons. In polymeric form, the molecular weight may be greater than1,000,000 daltons. The high molecular weight of TABM and theirnon-polar, hydrophobic nature have made the study of these moleculesvery difficult.

[0005] TABM are present in the serum at a concentration of between 10-50μg/ml. The monomeric units share some homology with the Ca chain of theT-cell receptor (TCR). However, TABM are not soluble forms of TCR andexhibit physiological and biochemical properties distinct from TCR.

[0006] There appears to be several types of TABM with differentfunctions. One particularly important function is their involvement inimmuno-regulation. For example, some TABM initiate delayed typehypersensitivity while other types appear to inhibit cell-mediatedimmunity. The latter type of TABM are associated with cytokines and inparticular interleukin 10 (IL-10) and transforming growth factor beta(TGF-β). It is thought that these TABM “focus” the cytokines to wherethe antigen is localised to suppress the cell-mediated immune responseto the antigen.

[0007] It is likely, therefore, that TABM are involved in a number ofclinical disorders or other physiological conditions involvingsuppression or activation of immune processes. However, as TABM arepresent in serum in only minute amounts, it has hithertofore beenimpractical to measure TABM levels or to use TABM in diagnosticprotocols.

[0008] In work leading up to the present invention, the inventors soughtmonoclonal antibodies to TABM. The isolation of such monoclonalantibodies enables the purification of large quantities of TABM and thedevelopment of diagnostic assays based on identification of specificTABM or amounts of specific TABM as well as the development oftherapeutic protocols based on modulating TABM levels.

SUMMARY OF THE INVENTION

[0009] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising” will be understood to imply the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers.

[0010] One aspect of the present invention provides animmuno-interactive molecule comprising a portion which is capable ofspecifally interacting with TABM.

[0011] Another aspect of the present invention is directed to ahybridoma cell line producing a monoclonal antibody comprising a bindingportion specific to TABM.

[0012] Yet another aspect of the present invention contemplates ahybridoma cell line having the characteristics of cell line MG3C9producing a monoclonal antibody capable of interacting with TABM.

[0013] Still yet another aspect of the present invention contemplates amethod of detecting TABM in a biological sample from a subject saidmethod comprising contacting said biological sample with animmunointeractive molecule specific for TABM or their derivatives orhomologues for a time and under conditions sufficient for animmunointeractive molecules-TABM complex to form and then detecting saidcomplex.

[0014] Another aspect of the present invention provides a compositioncomprising an immunointeractive molecules specific for TABM.

[0015] Yet another aspect of the present invention contemplates a methodfor determining assessing or otherwise monitoring the immunologicalstatus of an individual said method comprising screening for thepressure or level of TABM in a biological sample from said individual.

BRIEF DESCRIPTION OF THE FIGURES

[0016]FIG. 1 is a graphical representation of an ELISA of Cohn FractionIII TABM. One hundred ng of Cohn Fraction III TABM were coated tomicrotiter trays and assayed with dilutions of A) rabbit anti-human TABMR28. anti-immunoglobulin κ chain, λ chain or albumin:

[0017] B) monoclonal anti-human TcR α chain, β chain and (controls):anti-murine LyT-2, anti-H-2K:

[0018] C) affinity purified anti-TGF-β1, TGF-β2, TGF-β3. Bound antibodywas developed with alkaline phosphatase-conjugated to anti-murine orrabbit IgG.

[0019]FIG. 2 is a photographic representation showing resolution of CohnFraction III TABM. Purified TABM (FIG. 1) were reduced with 5% v/vβ2-mercaptoethanol and 400 ng resolved by SDS-PAGE in an 10-15% w/vpolyacrylamide gradient gel. Proteins were visualized by silver stain.Molecular weights were determined by the mobility of pre-stainedstandard proteins.

[0020]FIG. 3 is a graphical representation showing that monoclonalantibody MG3C9-1A12 binds TABM. Microtiter trays were coated with 500 ngTABM, IgG or IgM, and 100 μl 1:300 rabbit anti-TABM (R30), or 1:100culture supernatant from clone MG3C9-1A12 or 2B3 was added to the wells.Bound antibodies were developed with alkaline phosphatase-conjugatedgoat anti-rabbit or mouse IgG.

[0021]FIG. 4 is a graphical representation of the titration ofmonoclonal antibodies 3C9 and subclone MG3C9-1A12. Microtiter trays werecoated with 5-500 ng TABM/well and dilutions of antibody, culturesupernatants or ascites added to the wells. Bound antibody was developedwith peroxidase-conjugated goat anti-mouse IgG. A) Activity ofpre-cloning “parent” hybridoma MG3C9 and subclones.

[0022] B) Activity of MG3C9-1A12 culture supernatant, protein G-purifiedantibody and ascitic fluid. The curves show overlap, because “all”antibody was isolated by protein G, then taking into account theconcentration of the purified antibody, the curves overlapped.

[0023]FIG. 4C is a graphical representation of dilutions of humanImmunoglobulin negative serum coated to microtiter trays and thenassayed for TABM using MG3C9-1A12 (1:1000). The serem has a TABM “titer”of >256.000 and no immunoglobulin (Ig-,Ig) was detected in this serem.

[0024]FIG. 5 is a graphical representation showing that BA-Specific,MG3C9-1A12 TABM bear TcRα epitopes. Serum from a patient with a hightiter of TABM to BA-HSA individual was absorbed to MG3C9-1A12-sepharosebeads. The beads were eluted, and the eluate proteins absorbed toBA-HSA-sepharose beads. The eluted proteins were coated to microtiterwells at 200 ng/well. Monoclonal MG3C9-1A12, anti-TcR Cα-1, Cβ-1, orCδ-1 (1:100) was added to the wells, and bound antibody detected withalkaline phosphatase-conjugated goat anti-mouse IgG.

[0025]FIG. 6 is a graphical representation showing the MG3C9-1A12antibody detects TABM in human and primate serum. Human (Cohn FractionIII) TABM (hTABM) or 100 μl of dilutions of fetal calf serum (FCS),cynomologous monkey or sheep and human serum were coated to wells ofmicrotiter trays. A 1.1000 dilution of protein G-purified monoclonalMG3C9-1A12 was added, and bound antibody detected withperoxidase-conjugated sheep anti-mouse IgG.

[0026]FIG. 7 is a photographic representation of immunoblotting of TABMand serum with monoclonal MG3C9-1A12. Two hundred fifty μl of a 1:50dilution of normal human serum, and 15 μg of Cohn Fraction III TABM werereduced with 5% v/v β2-mercaptoethanol, and 4 μl of a 1:50 dilution ofserum or 240 ng TABM were resolved in an 8-25% w/v polyacrylamide gel.Proteins in the electrophoresed gel was transferred to an immobilonmembrane and blotted with 10 ml of a 1:1000 dilution of proteinG-purified monoclonal MG3C9- 1A12. Molecular weights were determined bythe mobilities of pre-stained standard proteins.

[0027]FIG. 8 is a photographic representation of a resolution of Jurkatproteins bound by Monoclonal MG3C9-1A12. 1×10⁷ Jurkat (T) or A1 (B)cells were lysed with 1 ml 1.0% v/v Triton X-100. The lysate wasabsorbed with 200 μl monoclonal MG3C9-1A12-sepharose beads. The washedbeads were eluted with 0.5 ml SDS-PAGE sample buffer and reduced by theaddition of 5% v/v β₂-mercaptoethanol. Four μl of the eluate wasresolved in an 8-25% v/v polyacrylamide gradient. 1:Jurkat. 2: A1proteins are silver stained. Molecular weights were determined by themobilities of pre-stained molecular weight standards.

[0028]FIG. 8B is a graphical representation of detergent lysates of thehuman T-cell line Jurkat and the B-cell (leukaemia) line A-1 absorberedto MG3C9-sepharose and the column eluted with 0.1M NaCO₃. The eluateswere used to coat microtiter trays and assayed with MG3C9-1A12. TheB-cell line is negative for TABM.

[0029]FIG. 9 is a graphical representation showing the presence of TABMin different fractions. Patient serum and different TABM, preparationswere serially diluted and plated onto ELISA plates. The amount of TABMwas determined using the mouse monoclonal anti-human TABM antibody(MG3C9-1A12).

[0030]FIG. 10 is a graphical representation showing the determination ofTABM and IgG levels in different fractions. Different amounts of TABMwere coated directly onto ELISA plates and (A) the presence of TABM wasdetected using the monoclonal anti-human TABM antibody. The presence ofIgG (B) was detected using a peroxidase labelled sheep anti-human IgG.

[0031]FIG. 11 is a graphical representation of the determination ofantigen specific IgG in various samples to different haptens. Varioushaptens were coated onto ELISA plates at 250 ng/well. The patient serum(A), MG3C9-1A12+ve TABM (B) and the purified BA-HSA specific TABM,BA-TABM (C) were serially diluted and tested for IgG to the haptens.

[0032]FIG. 12 is a graphical representation showing detection of antigenspecific TABM in different fractions. HSA (A) and BA-TABM (B) werecoated onto ELISA plates. The patient serum and TABM preparations wereserially diluted and tested for the presence of antigen specific TABMusing the mouse monoclonal anti-human TABM antibody (MG3C9-1A12).

[0033]FIG. 13 is a graphical representation of competitive inhibitionELISA between BA-TABM and various haptens. Purified BA-TABM was dilutedand pre-mixed with various haptens. The mixture was put onto an ELISAplate coated with the hapten BA-HSA and incubated. The amount of TABMbinding was determined using the mouse monoclonal anti-human TABMantibody (MG3C9-1A12).

COMPETITOR

[0034]FIG. 14 is a graphical representation showing competitiveinhibition ELISA between BA-TABM and various chemicals. Purified BA-TABMwas diluted and pre-mixed with various chemicals. The mixture was putonto an ELISA plate coated with the hapten BA-HSA and incubated. Theamount of TABM binding was determined using the mouse monoclonalanti-human TABM antibody (MG3C9-1A12).

COMPETITOR

[0035]FIG. 15 is a graphical representation of the determination of theinteraction of various TABM preparations and a panel of haptens. A panelof haptens were coated onto ELISA plates. Various samples andpreparations of TABM (A) PBS. (B) patient serum, (C) MG3C9-1A12+ve TABMand (D) BA-TABM were diluted and tested for the presence of haptenspecific TABM using the mouse monoclonal anti-human TABM antibody(MG3C9-1A12).

[0036]FIG. 16 is a graphical representation showing patient recall ofprose (immediate), tested pre and post toluene exposure, p=0.0003.

[0037]FIG. 17 is a graphical representation showing patient recall ofprose (delayed), tested pre and post toluene exposure, p=0.0009.

[0038]FIG. 18 is a graphical representation showing digit symbolrecalled by patients pre and post toluene exposure, p=0.0099.

[0039]FIG. 19 is a graphical representation showing letter cancellationby patients pre and post toluene exposure, p=0.038.

[0040]FIG. 20 is a graphical representation showing serum levels ofantigen specific IgG to BA-HSA, p=0.535.

[0041]FIG. 21 is a graphical representation of serum levels of antigenspecific TABM to BA-HSA. P=0.002.

[0042]FIG. 22 is a graphical representation of TABM levels to BA-HSAversus change in focal length in patients.

[0043]FIG. 23 is a graphical representation of antigen specific TABM toBA-HSA in patients and controls. Group 1:14-25 years Group 2:up to 13years duration of chemical exposure.

[0044]FIG. 24 is a graphical representation showing that increasinglevels of free BA-HSA bind to serum TABM and inhibit the specificbinding to plate bound BA-HSA.

[0045]FIG. 25 is a graphical representation of the levels of antigenspecific TABM to Candida albicans CTAB (A) and PEAT (B) mannans and tothe Hollister-Stier skin test preparation (C) was determined in acontrol and a patient group. P values are 0.0291, 0.0721 and 0.2166,respectively.

[0046]FIG. 26 is a graphical representation of the levels of antigenspecific IgG to Candida albicans CTAB (A) and PEAT (B) mannans and tothe Hollister-Stier skin test preparation (C) was determined in acontrol and a patient group. P values are 0.0458, 0.0303 and 0.0791respectively.

[0047]FIG. 27 is a graphical representation of the levels of TABM wereplotted against the levels of IgG for CTAB, PEAT and Hollister-StierCandida antigens.

[0048]FIG. 28 is a graphical representation of the levels of antigenspecific TABM (A) and IgG (B) to CTAB mannan are plotted against thenumber of episodes of thrush experienced within twelve months in thepatient group.

[0049]FIG. 29 is a graphical representation of the levels of antigenspecific TABM (A) and IgG (B) to CTAB mannan are plotted against thenumber of years with recurrent thrush in the patient group.

[0050]FIG. 30 is a graphical representation showing serum levels ofantigen-specific IgG to β-lactoglobulin (BLG), α-lactalbumin (AL) andα-s-casein (CA). Group 1=controls, Group 2=patients.

[0051]FIG. 31 is a graphical representation showing titration forβ-lactoglobulin-specific TABM. Dilutions of serum from a milk-intolerantpatient and purified BL-TABM were added to microtiter wells coated with1 μg/well β-lactoglobulin. After incubation and washing, rabbitanti-human TABM (1:300) was added, and bound antibodies detected withperoxidase-conjugated sheep anti-rabbit IgG.

[0052]FIG. 32 is a graphical representation showing serum levels ofantigen-specific TABM to (A)BLG, (B)AL, and (C)CA. Sera were diluted 1:4and 100 μl tested against 500 ng of antigen and detected as in FIG. 31.Group 1=controls, Group 2=patients.

[0053]FIG. 33 is a graphical representation of purified BL-TABM testedfor binding to gelatin, bovine serum albumin (BSA) and BLG. Serum from amilk-intolerant patient was absorbed with β-lactoglobulin-conjugatedsepharose beads, and the beads eluted with NaCO₃. The dialyzed eluatewas added to microtiter wells coated with 5 μg β-lactoglobulin, BSA orgelatin. Bound protein was detected with rabbit anti-human TABM, andperoxidase-conjugated sheep anti-rabbit IgG.

[0054]FIG. 34 is a photographic representation depicting resolution ofβ-lactoglobulin specific TABM by SDS-PAGE. Eight hundred and seventy ngreduced or non-reduced BL-TABM were resolved by SDS-PAGE in a 10-15% w/vgradient. Reduced proteins were visualized by silver stain. Apparentmolecular weights were determined by the mobility of molecular weightstandards. A) non-reduced; B) reduced.

[0055]FIG. 35A is a graphical representation showing increasing levelsof β-lactoglobulin specific TABM (BL-TABM) induce an increasing amountof TNF-α by normal peripheral blood mononuclear cells. The addition ofβ-lactoglobulin enhances this effect further.

[0056]FIG. 35B is a graphical representation showing that LPS (a stronginducer of TNF-α) combined with BL-TABM and β-lactoglobulin all combineto potentiate the production of TNF-α by normal PBMNC.

[0057]FIG. 36 is a graphical representation showing detection of TGF-βin BL-TABM by ELISA. Ten to twenty μl BL-TABM were mixed with a 1:500dilution of rabbit anti-TGF-β1, 2 or 3, and the mixture added tomicrotiter wells coated with 10 ng corresponding TGF-β1, 2 or 3peptides. The inhibition of the binding of anti-TGF-β by BL-TABM wascompared to that obtained by immunogenic peptide to compute the amountof TGF-β in the TABM.

O.D. anti-TGF-β+BL-TABM ×100 O.D. anti-TGF-β

[0058] A) Inhibition of ELISA by TGF-β1 peptide.

[0059] B) Inhibition of ELISA by TGF-β2 peptide.

[0060] C) Quantity of TGF-β1 in BL-TABM based on ELISA inhibition.

[0061]FIG. 37 is a photographic representation of immunoblotting ofBL-TABM with anti-TGF-β antibodies. Eight hundred and seventy ngnon-reduced BL-TABM were resolved and transferred to immobilonmembranes. The milk-blocked membranes were incubated with rabbitanti-TGF-β2 antibodies, and bound antibody IgG and NBT-BCIP substrate.

[0062] The following abbreviations are used in the subjectspecification. Abbreviation Definition AL α-Lactoalbumin AG Antigencaptive ELISA BA Benzoic acid BA-TABM Benzoic acid specific TABM BL(BLG) β-Lactoglobulin BSA Bovine serum albumin BA-HSA Benzoic acidconjugated human serum albumin CA α-s-Casein CTAB Cetryl trimethylammonium bromide ELISA Enzyme linked immunosorbant assay ES Electricalstimulate F-HSA (Form-HSA) Formaldehyde conjugated human serum albuminFCS Fetal calf serum FCSi Heat inactivated FCS hTABM Human TABM HSAHuman serum albumin HS Horse serum HSA-TABM HSA specific TABM MG3C9-1A12Anti human TABM monoclonal antibody M_(r) Molecular weight O.D. Opticaldensity PBMNC Peripheral blood mononuclear cells PBS Phosphate bufferedsaline PBS-Tw Phosphate buffered saline-Tween washing solution R28, R30Rabbit anti-human TABM sera SDS-PAGE Sodium dodecylsulphate-polyacrylamide gel electrophoresis SNP Sodium nitroprusside SPSubstance P TABM T-cell antigen binding molecules or T-cell derivedantigen binding molecules TCR T-cell receptor TCRα-1 Monoclonal antibodyto TCR α chain TMB 3′,3,5′,5-Tetramethyl benzidine TNF-α Tumour necrosisfactor-α TNP-BSA Trinitrophenol conjugated BSA Tw Tween

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0063] The present invention provides an isolated immunointeractivemolecule comprising a portion which is capable of specificallyinteracting with a TABM.

[0064] The TABM may be of animal or possibly avian origin. Preferredanimals are mammals such as humans, primates, livestock animals (eg.sheep, cows, horses, donkeys, pigs), laboratory test animals (eg.rabbits, mice, guinea pigs), companion animals (eg. dogs, cats) andcaptive wild animals (eg. deer, foxes, kangaroos). Most preferably, themammal is a human. Preferred avian species are chickens, ducks,ostriches, emus and caged birds. The immunointeractive molecule may bespecific to a particular TABM or may broadly interact with a range ofTABM. A particular TABM is conveniently defined by inter alia referenceto epitopes, cytokine interacting ability and/or antigen specificity.

[0065] The immunointeractive molecule of the present invention ispreferably an antibody and most preferably a monoclonal antibody or arecombinant, chemically synthetic, hybrid form, derivative or functionalequivalent thereof. A “derivative” in this context includes a fragment,portion, part, homologue or analogue of an antibody and, moreparticularly, a monoclonal antibody. Where the interactive molecule is amonoclonal antibody, it may be the entire monoclonal antibody or it maybe a TABM binding portion thereof or a hybrid between a carrier moleculeand a TABM binding portion of a TABM specific monoclonal antibody. Thepresent invention extends to polyclonal antibodies to TABM to the extentthat the polyclonal antibody has been rendered monovalent to thespecific TABM or has been otherwise derivatised. The present inventionalso extends to antibodies (monoclonal or polyclonal antibodies) tosynthetic peptides or peptide fragments of a TABM or corresponding to aportion of a TABM. Reference hereinafter to an “immunointeractivemolecule” specifically includes a monoclonal antibody as well as itsderivatives and novel forms of polyclonal antibodies or monovalentpolyclongal antibodies..

[0066] It is within the scope of this invention to include any secondinteractive molecules (eg. monoclonal or polyclonal antibodies orfragments of antibodies or synthetic antibodies) directed to the firstmentioned immunointeractive molecules referred to above. Both the firstand second immunointeractive molecules may be used in detection assays.Alternatively, the first immunointeractive molecuic may be used withcommercially available anti-immunoglobulin antibodies in diagnosticassays. An antibody as contemplated herein includes any antibodyspecific to any region of TABM. As stated above, the antibody may bespecific to a particular TABM or may broadly interact to all TABM or aclass of TABM.

[0067] Polyclonal and monoclonal antibodies are obtainable byimmunization with purified or substantially purified TABM and eithertype may be utilized in for immunoassays. The methods of obtaining bothtypes of sera are well known in the art. Polyclonal sera are lesspreferred but are relatively easily prepared by injection of a suitablelaboratory animal with an effective amount of TABM, or an antigenic partthereof, collecting serum from the animal and isolating specific sera byany of the known immunoadsorbent techniques. Although antibodiesproduced by this method are utilizable in virtually any type ofimmunoassay, they are generally less favoured because of the potentialheterogeneity of the product.

[0068] The use of monoclonal antibodies in an immunoassay isparticularly preferred because of the ability to produce them in largequantities and the homogeneity of the product. The preparation ofhybridoma cell lines for monoclonal antibody production derived byfusing an immortal cell line and lymphocytes sensitized against theimmunogenic preparation may be achieved by techniques which are wellknown to those who are skilled in the art. In one approach, a myelomacell line such as P3-NS1-Ag4-1 (NS-1) is fused with spleen cells from ananimal immunized with TABM. A convenient source of TABM for theimmunization is TABM purified from Cohn Fraction III (BaxterPharmaceutical). Alternatively, the TABM may be isolated from serum orother appropriate body fluid. A particularly useful hybridoma cellproduced in accordance with the present invention is designated hereinMG3C9 and produces monoclonal antibody MG3C9-1A12.

[0069] This aspect of the present invention is hereafter described withreference to one particularly useful means of producing animmunointeractive molecule in the form of a monoclonal antibody to TABM.

[0070] Human serum Cohn Fraction III is used as a source of TABM whichis purified on a sephacryl (eg. S-300) or sepharose (eg. 3C9) column.Rabbit anti-human TABM antisera is then prepared by immunising rabbitswith human serum TABM. The polyvalent antisera are specific for humanTABM produced by T-cells and do not bind human serum albumin, IgG, IgMor TGFβ1, 2 or 3 immunogenic peptides. Mice are then immunised with thepurified TABM and the spleens removed. Spleen cells are fused with theP3-NS1-Ag4-1 (NS1) myeloma cell line. Monoclonal antibody is thenscreened for by enzyme linked immunosorbent assay (ELISA). For example,TABM monoclonal secreting cells are identified by the monoclonalantibody binding to immobilized human TABM. Binding activity is detectedusing a labelled anti-immunoglobulin antibody preparation. Cells arethen cloned and grown to produce monoclonal antibody to TABM.

[0071] Accordingly, another aspect of the present invention provides ahybridoma cell line producing a monoclonal antibody comprising a bindingportion specific to TABM.

[0072] More particularly, the present invention is directed to ahybridoma cell line having the characteristics of cell line MG3C9producing a monoclonal antibody capable of interacting with TABM.Preferably, the monoclonal antibody is MG3C9-1A12.

[0073] Another aspect of the present invention contemplates a method fordetecting TABM in a biological sample from a subject said methodcomprising contacting said biological sample with an immunointeractivemolecule specific for TABM or their derivatives or homologues for a timeand under conditions sufficient for an immunointeractive molecule-TABMcomplex to form and then detecting said complex.

[0074] The presence of TABM may be accomplished in a number of ways suchas by Western blotting and ELISA procedures. A wide range of immunoassaytechniques are available as can be seen by reference to U.S. Pat. Nos.4,016,043, 4,424,279 and 4,018,653. These include both thenon-competitive assays as well as competitive binding assays. Theseassays also include direct binding of a labelled antibody to a target.

[0075] Sandwich assays are particularly useful. A number of variationsof the sandwich assay technique exist, and all are intended to beencompassed by the present invention. Briefly, in a typical forwardassay, an unlabeled immunointeractive molecule is immobilized on a solidsubstrate and the sample to be tested for the presence of a TABM isbrought into contact with the bound molecule. After a suitable period ofincubation, for a period of time sufficient to allow formation of animmunointeractive molecule-TABM complex, a second immunointeractivemolecule specific to the TABM, labelled with a reporter molecule capableof producing a detectable signal is then added and allowed to incubate,allowing time sufficient for the formation of another complex ofimmunointeractive molecule-TABM-labelled interactive molecule. Anyunreacted material is washed away, and the presence of the TABM isdetermined by observation of a signal produced by the reporter molecule.The results may either be qualitative, by simple observation of thevisible signal, or may be quantitated by comparing with a control samplecontaining known amounts of TABM. Variations on the forward assayinclude a simultaneous assay, in which both sample and labelled antibodyare added simultaneously to the bound antibody. In another variation,the first immunointeractive molecule is a polyclonal antibody and thesecond, labelled interactive molecule is TABM-specific monoclonalantibody. These techniques are well known to those skilled in the art,including any minor variations as will be readily apparent.

[0076] A particularly useful variation permits the identification of aTABM specific interactive molecule. In this assay, purified TABM areimmobilized to a solid support such as a microtitre well. A sample,putatively carrying a TABM immunointeractive molecule, is brought intocontact with the immobilized TABM. If present, the immunointeractivemolecule binds to the immobilized TABM. An anti-immunoglobulin antibodypreparation, labelled with a reporter molecule capable of providing anidentifiable signal, is the then used to identify bound TABM-specificimmunointeractive molecules.

[0077] In accordance with the present invention the sample is one whichmight contain TABM (or a TABM specific immunointeractive molecule)including serum, whole blood, cell extract, tissue biopsy, saliva,mucosal secretions, lymph, tissue fluid and respiratory fluid. Thesample is, therefore, generally a biological sample comprisingbiological fluid but also extends to fermentation fluid and supernatantfluid such as from a cell culture.

[0078] In the typical forward sandwich assay, a first immunointeractivemolecule having specificity for the TABM or antigenic parts thereof, iseither covalently or passively bound to a solid surface. The solidsurface is typically glass or a polymer, the most commonly used polymersbeing cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene. The solid supports may be in the form of tubes, beads,discs or microplates, or any other surface suitable for conducting animmunoassay. The binding processes are well-known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing. The polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated for a period of time sufficient (e.g.2-40 minutes or overnight if more convenient) and under suitableconditions (e.g. from room temperature to about 40° C. including fromabout 25° C. to about 37° C.) to allow binding of any subunit present inthe antibody. Following the incubation period, the solid phase is washedand dried and incubated with a second immunointeractive moleculespecific for a portion of the TABM. The second immunointeractivemolecule is linked to a reporter molecule which is used to indicate thebinding of the second immunointeractive molecule to the TABM. Similarconsiderations apply after screening for a TABM-specificimmunointeractive molecule.

[0079] An alternative method involves immobilizing the target TABMmolecules in the biological sample and then exposing the immobilizedtarget to specific immunointeractive molecule which may or may not belabelled with a reporter molecule. Depending on the amount of target andthe strength of the reporter molecule signal, a bound target may bedetectable by direct labelling with the antibody. Alternatively, asecond labelled immunointeractive molecule, specific to the firstimmunointeractive molecule is exposed to the TABM-firstimmunointeractive molecule complex to form a TABM-firstimmunointeractive molecule-second immunointeractive molecule tertiarycomplex. The complex is detected by the signal emitted by the reportermolecule.

[0080] A particularly preferred alternative method is to immobiliseantigen on a solid support and then bring into contact the biologicalsample containing the putative TABM. The antigen is used, therefore, totrap the specific TABM. An anti-TABM antibody is then used to detectbound TABM either directly if labelled with a reporter molecule orindirectly via a labelled anti-immunoglobulin antibody.

[0081] By “reporter molecule” as used in the present specification, ismeant a molecule which, by its chemical nature, provides an analyticallyidentifiable signal which allows the detection of antigen-boundantibody. Detection may be either qualitative or quantitative. The mostcommonly used reporter molecules in this type of assay are eitherenzymes, fluorophores or radionuclide containing molecules (i.e.radioisotopes) and chemiluminescent molecules. Biotin andAvidin/Streptavidin may also be employed.

[0082] In the case of an enzyme immunoassay, an enzyme is conjugated tothe second antibody, generally by means of glutaraldehyde or periodate.As will be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, beta-galactosidase and alkaline phosphatase, amongst others.The substrates to be used with the specific enzymes are generally chosenfor the production, upon hydrolysis by the corresponding enzyme, of adetectable colour change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labeledimmunointeractive molecule is added to the first immunointeractivemolecule-TABM complex, allowed to bind, and then the excess reagent iswashed away. A solution containing the appropriate substrate is thenadded to the complex of immunointeractivemolecule-TABM-immunointeractive molecule. The substrate will react withthe enzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of TABMwhich was present in the sample. The term “reporter molecule” alsoextends to use of cell agglutination or inhibition of agglutination suchas red blood cells on latex beads and the like.

[0083] Alternately, fluorescent compounds, such as fluorescein andrhodamine, may be chemically coupled to antibodies without alteringtheir binding capacity. When activated by illumination with light of aparticular wavelength, the fluorochrome-labelled immunointeractivemolecule adsorbs the light energy, inducing a state to excitability inthe molecule, followed by emission of the light at a characteristiccolour visually detectable with a light microscope. As in the ELISA, thefluorescent labelled immunointeractive molecule is allowed to bind tothe first immunointeractive molecule-TABM complex. After washing off theunbound reagent, the remaining tertiary complex is then exposed to thelight of the appropriate wavelength the fluorescence observed indicatesthe presence of the hapten of interest. Immunofluorescene and ELISAtechniques are both very well established in the art and areparticularly preferred for the present method. However, other reportermolecules, such as radioisotope, chemiluminescent or bioluminescentmolecules, may also be employed.

[0084] The assays of the present invention are sensitive and can be usedto detect nanogram amounts of TABM.

[0085] The present invention further extends to genetic assays for TABM.For example, anti-TABM antibodies may be used to screen expressionlibraries for genetic sequences encoding all or antigenic parts of TABM.This permits a ready source of recombinant TABM for diagnostic andtherapeutic purposes and also provides genetic sequences such as primersand probes to screen an individual for mRNA specificity for a TABM geneor potential aberrations in a TABM gene. Accordingly, the presentinvention extends to nucleic acid molecules encoding a TABM ashereinbefore defined as well as genetic assays for TABM genes.

[0086] As stated above, the present invention extends to derivatives ofthe immunointeractive molecules. Derivatives include single or multipleamino acid substitutions, deletions and/or additions to theimmunointeractive molecules. “Additions” to amino acid sequences includefusions with other peptides, polypeptides or proteins or fusions tonucleotide sequences.

[0087] Analogues of the immunointeractive molecule contemplated hereininclude, but are not limited to, modification to side chains,incorporating of unnatural amino acids and/or their derivatives duringpeptide, polypeptide or protein synthesis and the use of crosslinkersand other methods which impose conformational constraints on theproteinaceous molecule or their analogues. These types of modificationsare useful in stabilizing the immunointeractive molecules for use indiagnostic assays or in therapeutic protocols.

[0088] Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate:trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzenesulphonic acid (TNBS); acylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5-phosphate followed by reduction with NaBH₄.

[0089] The guanidine group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

[0090] The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitisation, forexample, to a corresponding amide.

[0091] Sulphydryl groups may be modified by methods such ascarboxymethylation with iodoacetic acid or idoacetamide; performic acidoxidation to cysteic acid; formation of a mixed disulphides with otherthiol compounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuri-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

[0092] Tryptophan residues may be modified by, for example, oxidationwith N-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

[0093] Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

[0094] Examples of incorporating unnatural amino acids and derivativesduring peptide synthesis include, but are not limited to, use ofnorleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoicacid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,omithine, sarcosine. 4-amino-3-hydroxy-6-methylheptanoic acid. 2-thienylalanine and/or D-isomers of amino acids. A list of unnatural amino acid,contemplated herein is shown in Table 1.

[0095] Crosslinkers can be used, for example, to stabilise 3Dconformations, using homo-bifunctional crosslinkers such as thebifunctional imido esters having (CH₂)_(n) spacer groups with n=1 ton=6. glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctionalreagents which usually contain an amino-reactive moiety such asN-hydroxysuccinimide and another group specific-reactive moiety such asmaleimido or dithio moiety (SH) or carbodiimide (COOH). In addition.peptides can be conformationally constrained by, for example,incorporation of C_(α) and N_(β)-methylamino acids, introduction ofdouble bonds between C_(α) and C_(β) atoms of amino acids and theformation of cyclic peptides or analogues by introducing covalent bondssuch as forming an amide bond between the N and C termini, between twoside chains or between a side chain and the N or C terminus. TABLE INon-conventional Non-conventional amino acid Code amino acid Codeα-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrateMgabu L-N-methylarginine Nmarg aminocyclopropane- CproL-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmaspaminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- NorbL-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglucyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanineCpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine NmleuD-arginine Darg L-N-methyllysine Nmlys D-aspartic acid DaspL-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine NmnleD-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid DgluL-N-methylornithine Nmorn D-histidine Dhis L-N-methylphenylalanine NmpheD-isoleucine Dile L-N-methylproline Nmpro D-leucine DleuL-N-methylserine Nmser D-lysine Dlys L-N-methylthreonine NmthrD-methionine Dmet L-N-methyltrptophan Nmtrp D-ornithine DornL-N-methyltyrosine Nmtyr D-phenylalanine Dphe L-N-methylvaline NmvalD-proline Dpro L-N-methylethylglycine Nmetg D-serine DserL-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine NleD-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltrptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmry N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-merhylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-mehylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) NnbhmN-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycinecarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane

[0096] As stated above, these types of modifications may be important tostabilise the immunointeractive molecule if administered to anindividual or for use as a diagnostic reagent.

[0097] Other derivatives contemplated by the present invention include arange of glycosylation variants from a completely unglycosylatedmolecule to a modified glycosylated molecule. Altered glycosylationpatterns may result from expression of recombinant molecules indifferent host cells.

[0098] Another aspect of the present invention extends to theimmunointeractive molecule in composition form. Such compositions areparticularly useful as therapeutic compositions and may be referred toas pharmaceutical compositions. The compositions are useful incontrolling TABM activity by administering an immunointeractive moleculecapable of binding to the TABM and inhibiting or otherwise reducing theactivity of the TABM. This is particularly important where the TABM isinvolved in immune suppression or immune activation. Accordingly, thepresent invention contemplates a composition comprising animmunointeractive molecule specific for TABM.

[0099] The composition forms suitable for injectable use include sterileaqueous solutions (where water soluble) or sterile powders for theextemporaneous preparation of sterile injectable solutions. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or diluent containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The preventions of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thitmerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0100] Sterile injectable solutions are prepared by incorporating theimmunointeractive molecule in the required amount in the appropriatesolvent or diluent as followed by sterilization such as by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze-drying technique which yield a powder ofthe immunointeractive molecule plus any additional desired ingredientfrom previously sterile-filtered solution thereof.

[0101] Pharmaceutically acceptable carriers and/or diluents include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the immunointeractive molecule, use thereof in thetherapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

[0102] The principal immunointeractive molecule will be added in aconcentration effective to interact to a TABM and inhibit or reduce thefunction of the TABM. For example, an effective amount may range fromabout 10 ng to about 2000 ng, or 50 ng to about 1000 ng or 100 ng toabout 500 ng or other suitable effective amount. Of course, largeramounts may be administered such as from 10 μg to about 2000 mg. It issometimes more convenient to express dosage amounts in terms of kilogramper body weight. Accordingly, the effective amount may be from, forexample, about 0.5 ng/kg body weight to about 1000 ng/kg body weight oran amount there between.

[0103] Although not intending to limit the present invention to any onetheory or mode of action, the main function of TABM is likely to beimmuno-regulatory. In a murine model, for example, a rise in TABMaccompanies the humoral response. In most studies to date, the functionof TABM has been suppression of the cell-mediated response to specificantigens. Particularly high levels of TABM have been reported inexperimentally induced immune deviation, indicating a Th2 response withsuppression of the Th1 response.

[0104] TABM are associated with cytokines, in particular TGF-β andIL-10. As is exemplified herein, the inventors have purified TABMspecific to β-lactoglobulin (BL), from a patient intolerant to cow'smilk, and TABM specific to benzoic acid conjugated to human serumalbumin (BA-HSA) from another patient sensitive to the solvent toluene.In both cases, the presence of TGF-β has been detected.

[0105] It is proposed herein that TABM act by focusing cytokines such asTGF-β to sites where the antigen is localised TGF-β is a potentinhibitor of cell mediated immunity and increased local concentrationsof this cytokine would inhibit the development of a cell mediated immuneresponse to the antigen. Most of the TGF-β associated with TABM ispresent in an inactive form. When TABM bind to antigen, the TGF-βbecomes activated. This seems to occur optimally at a relatively lowantigen concentration.

[0106] In addition, elevated levels of total TABM are detected, inaccordance with the present invention, in patients symptomatic with AIDSand antigen specific TABM in individuals with chronic filiariasis orasymptomatic carriers of the filiarial worm. In both these disorders,there is suppressed cell mediated immunity to the infectious agent.

[0107] The inventors have performed clinical studies, measuring levelsof antigen specific TABM.

[0108] In a study of milk intolerant patients, the inventors foundraised levels of TABM to one or more of the milk proteins,β-lactoglobulin, α-lactalbumin and/or α-s-casein, in the patient groupwhen compared to controls.

[0109] In another study, the inventors found elevated levels of TABM toBA-HSA in patients sensitive to the solvent toluene. This antigen wasthought to be suitable as benzoic acid is a major metabolite of toluene.

[0110] TABM may also bind to drug, drug-protein conjugates or theirmetabolites and have a role in drug mediated allergies.

[0111] The immunointeractive molecule of the present invention was alsoused to measure mannan specific TABM in a study of women susceptible tothrush. Thrush was used as a model as the literature indicates that cellmediated immunity is suppressed. The inventors found elevated levels ofTABM specific to Candida albicans mannan in these patients.

[0112] It is known that certain cytokines enhance the release ofneuropeptides from sensory nerves. This has been extensively studied inthe case of Interleukin-1. The inventors have now found that TGF-β alsoenhances the release of neuropeptides from sensory nerves. This effectis dose dependent and lost when animals are pre-treated with Capsaicin.The effect is blocked by anti TGF-β antibody.

[0113] The BA-TABM also enhances the release of neuropeptides in a dosedependent manner. This effect is blocked by anti-TGF-β antibody and isattributable to the co-presence of TGF-β in the TABM preparation. Theaddition of antigen (BA-HSA) to the TABM may enhance or inhibitneuropeptide release: there is enhancement at low concentrations ofBA-HSA and inhibition at higher concentrations. This seems consistentwith the effect of antigen concentration in activating TGF-β in theTABM.

[0114] TABM detectable with the immunointeractive molecule of thepresent invention indicates immune deviation with suppression of thecell mediated immune response to specific antigens. TABM levels mayprovide an index of the balance between Th1 and Th2 immune responses.This could be important in assessing the immunological status in cancer,autoimmunity and infectious diseases. Also, the presence of TABM mayindicate a continuing immune response with a failure to achieve immunetolerance to non replicating antigens, for example to common foodproteins. TABM specific to such proteins may indicate “low dosetolerance” and not anergy or deletion.

[0115] As an example, the measurement of TABM specific to melanomaantigens may be helpful in evaluating the cell mediated immune responseto this type of tumour. An impairment of the response could indicate arisk of dissemination. A similar measurement strategy may be importantin other cancers where the balance between cell mediated and humoralimmunity is pivotal in controlling the tumour growth.

[0116] TGF-β is reported as modulating certain parasitic diseases,particularly by limiting tissue damage. The inventors have identifiedTABM in the sera of patients who are chronic carriers of filiariasis.TGFβ is reported to inhibit cell mediated immunity in schistosomiasis,sleeping sickness and leishmaniasis. Measurement of TABM in suchinfections may be helpful in evaluating resistance to infection and thesusceptibility to tissue damage.

[0117] TABM specific to viral peptides are present in patients withAIDS. This may indicate a decline in cell mediated immune response tothe virus. Similar measurements may be helpful in patients with serumhepatitis who have become carriers, where there is thought to be asimilar abnormality in the immune response to the virus. Also it ispossible that serial measurement of total TABM may be of assistance inassessing the progress of AIDS.

[0118] In mycobacterial infections, for example tuberculosis andleprosy, the immune defence is critically dependent on the cell mediatedimmune response. If TABM levels to mycobacterial antigens are raisedthis may indicate a vulnerability to these infections. Measurement ofTABM to antigens of Mycobacterium leprae may help distinguish betweentuberculoid and lepromatous leprosy.

[0119] As indicated above, an immune response with production of TABM tofood proteins or simple chemicals could be associated with persistentsymptoms. TABM specific to environmental antigens could be important inmany cases of food intolerance, which can cause functional bowelsymptoms, or chemical sensitivity. Assays of TABM for chemical antigensmay be helpful in evaluating at risk personnel who are occupationallyexposed to chemicals. Similar assays could be of assistance inevaluating drug reactions and the possible adverse effects of siliconimplants.

[0120] In some cases it would be helpful to determine the immuneresponse produced by vaccination. For example in the case oftuberculosis it is preferable that a Th1 immune response is induced. Themeasurement of TABM specific to antigens used for vaccination mayindicate the outcome of the vaccination procedure i.e. whether apredominantly Th1 or Th2 response has been achieved.

[0121] In certain “auto-immune” disorders such as multiple sclerosis,Th1 immune responses may be directed to self antigens. Therapy can beassociated with a shift in the response to the Th2 mode. For examplethis may be the outcome when patients with multiple sclerosis aretreated with Interferon. Serial measurement of TABM to the auto antigensmay help monitor the effectiveness of treatment.

[0122] Accordingly, the present invention further contemplates a methodfor assessing, monitoring or diagnosing a disease or other physiologicalcondition by determining the levels of totatl or antigen-specific TABMin an individual.

[0123] In a further embodiment, an individuals own TABM may be purifiedand the readministration to the same person or a related person.Accordingly, the antibodies to TABM have a particularly useful role inpurifying TABM for potential clinical use.

[0124] Assaying the level of TABM and therapy involving TABM may also beimportant in allograft rejection and in the regulation of immuneresponses.

[0125] For example, a excess Th1 response has been associated withmiscarriage and spontaneous abortion. The immune response may beregulatable to protect the developing foetus such that the foetus doesnot provoke an unwanted immune response. TABM and assaying TABM levelsare important in this respect and are encompassed by the presentinvention.

[0126] Accordingly, assays for TABM levels are important for a range ofdiagnoses including the detecting of microbial and parasitic infection,cancer, neuropeptide release and allergies including drug, milk and foodallergies.

[0127] The present invention is further described by the followingnon-limiting Examples.

Examples 1-19 relate to the production and characteristics of amonoclonal antibody to TABM EXAMPLE 1 TABM

[0128] Five grams of human serum Cohn Fraction III (BaxterPharmaceuticals) was mixed with 10 ml 2M urea in phosphate-bufferedsaline (PBS, pH 7.2), and after 15 min agitation, the mixture wascentrifuged for 30 min at 9,000 rpm. The supernatant was retained andloaded on a 3.5 cm×67 cm column containing sephacryl S-300 equilibratedin the same buffer. The column was pumped at 950 ml/hr and 5 ml columnfractions were collected. Based on the predetermined chromatographicpositions of IgM. IgG, human albumin and ovalbumin, the column fractionswere pooled as Fraction I, void volume (>MW 600,000), Fraction II. (MW10⁵−3×10⁵), Fraction III, (MW 4×10⁴−1×10⁵), Fraction IV (MW <3×10⁴).Fractions containing TABM (see Table 1) were pooled and precipitated bythe addition of (NH₄)₂SO₄ to a final concentration of 43%, as described(2). The precipitated proteins were solubilized in PBS and then absorbedwith sepharose 4B beads conjugated with anti-human Ig (whole molecule,Sigma) and anti-human albumin (Sigma, St. Louis, Mo.).

EXAMPLE 2 Antisera/Antibodies

[0129] Rabbit anti-human TABM sera (R28, R30) were prepared as describedby immunizing rabbits with human serum TABM prepared by chromatographyin carboxymethyl cellulose and immunoabsorption. The antisera arespecific for human TABM produced by T lymphocytes and do not bind humanalbumin, human IgG, IgM or TGF-β1, 2, or 3 immunogenic peptides.Monoclonal antibodies to TcRα (TcRα-1) and beta chains (TcRβ- 1) werepurchased from T-Cell Diagnosis (Woburn, Mass.) and T Cell Sciences(Cambridge, Mass.), respectively. Rabbit anti-mouse IgG and IgM (JacksonImmunoresearch Lab, Inc.) was used to isotype the monoclonal antibodies.

EXAMPLE 3 Immunisation of Mice with Human TABM

[0130] Female BALB/c mice. 6-8 weeks old were immunizedintraperitoneally (i.p.) with 5-10 μg of purified human TABM in completeFreund's adjuvant (Sigma Chemical Company). A second i.p. injection wasgiven with Freund's incomplete adjuvant (Sigma Chemical Company) onemonth later. The third and fourth immunisations were given in a solutionof Poly A and Poly U (equal volumes of 1 mg/ml of each) at 3 and 7months respectively. Four days before the fusion a fifth i.p.immunisation in PolyA:Poly U was given.

EXAMPLE 4 Spleen Cell Preparation

[0131] The spleen was removed and placed in serum free RPMI-1640,followed by two rinses. Spleen cells were teased out, collected into atube and larger clumps allowed to settle. Only the supernatantcontaining single cells were collected. The cells were washed twice inserum free RPMI-1640 culture medium. Cell viability was near 100%.

EXAMPLE 5 Myeloma Cells

[0132] The P3-NS1-Ag4-1 (NS-1) myeloma cell line was used and grown inRPMI-1640 containing 10% v/v heat inactivated (56° C., 30 min) fetalcalf serum (FCS). Cells in logarithmic growth were centrifuged andresuspended in 45 ml serum free medium. The cells were washed twice inserum free medium and a cell viability count made and found to be >95%viable.

EXAMPLE 6 Fusion

[0133] The appropriate number of spleen and myeloma cells were addedtogether, mixed well and centrifuged (third wash), thoroughly drainingthe supernatant. Cell fusion was carried out essentially as outline byKohler and Milstein (1) with the following changes: a 50% w/v solutionof PEG 4000, (Gibco BRL, N.Y.) was used, the red blood cells in thespleen cell preparation were not lysed, the number of spleen to myelomacells was 20×10⁶: 55×10⁶ (approximately 2:1). The selection medium(RPMI-1640 15% v/v bovine serum (FCS)+HAT was added at 24 hr. The mediumalso contained 10% v/v of P338 (IL-6) conditioned medium. Dividing cellswere passaged into RPMI-1640 15% v/v FCSi HT without IL-6 conditionedmedium, then into RPMI-1640 15% v/v FCSi. Subsequently cells wereadapted to culture in RPMI-1640 10% v/v FCSi.

EXAMPLE 7 Screening for Monoclonal Antibody

[0134] The screening of fused cells for the production of specificantibody was carried out using the enzyme linked immunosorbent assay(ELISA). A 96 well ELISA plate (Costar, Cambridge, Mass.) was coatedwith purified human TABM at 500 ng/well in 0.06M carbonate buffer. pH9.5 and incubated overnight at room temperature. The plate was washed 5times with PBS-0.05% v/v Tween-20 (PBS-Tw) and 50-100 μl of culturesupernatant from individual wells with hybrids was added. The sampleswere incubated at 37° C. for 90 mins, followed by washing with PBS-Tw. Aperoxidase conjugated sheep anti-mouse immunoglobulins (Silenus,Australia) was diluted {fraction (1/2000)} in PBS-Tw-1% w/v gelatin and100 μl placed per well. Incubation was carried out at 37° C. for 90rains, followed by washing. The reaction was developed with3′,3,5′,5-tetramethyl benzidine (TMB) substrate (KPL, MA) at roomtemperature until an OD of approximately 2 was obtained. The reactionwas stopped with 2M H₂SO₄ and the plate OD read at 450 nm.

EXAMPLE 8 Cloning of Hybridoma Cells

[0135] The cloning of a positive “parent” hybrid was carried out by thelimiting cell dilution assay. Cells were diluted so that on cell in 200μl of RPMI 1640-15% v/v FCSi were plated per well of 96 well flat bottomtissue culture plate (NUNC, Denmark). After 4-5 days, wells werescreened visually to identify those wells with a single clone. Atapproximately 10 days culture, supernatants were screened by ELISA toidentify positive antibody producing clones. Most wells were screenedtwice for antibody activity.

EXAMPLE 9 Production of Monoclonal Antibody in Culture Supernatant

[0136] Cloned hybridoma cells (10-20 ml) in logarithmic growth wereadded to 50-60 ml RPMI 1640-15% v/v FCSi culture medium in 500 ml glassbottles. After 1-2 days an equal volume of serum free medium was addedand the cells allowed to row to death (5-6 days). The culturesupernatant was collected by centrifugation and stored at 4° C.

EXAMPLE 10 Production of Monoclonal Antibody in Ascites

[0137] Eight week old female BALB-c mice were injected i.p. with 0.5 mlof pristane (Sigma Chemical Company). Six days later, mice were injectedi.p. with 3.75×10⁶ cloned cells in serum free culture medium. Asciteswas collected 20 days later, centrifuged to remove cells and pristaneand stored frozen.

EXAMPLE 11 Isotyping of the Anti-human TABM Monoclonal Antibody

[0138] An ELISA with similar conditions as above was carried out usingrabbit anti-mouse IgG and rabbit anti-mouse IgM antibodies (JacksonImmunoresearch Lab, Inc, USA). The enzyme conjugate was peroxidaseconjugated sheep anti-rabbit immunoglobulins (Silenus, Australia).

EXAMPLE 12 Specificity of the Anti-human TABM Monoclonal Antibody

[0139] ELISA plates were coated at 500 ng/well with various proteinswhich included: Human serum albumin (Calbiochem, Calif.), bovine serumalbumin, beta-lactoglobulin, casein, (Sigma Chemical Company),“Intragam”-human immunoglobulin (CSL, Melbourne), and human TABM as apositive control. After binding, a {fraction (1/2000)} dilution ofpurified mouse monoclonal antibody to human TABM was added and incubatedat 37C. for 90 minutes. After washing, a peroxidase labelled sheepanti-mouse was added.

EXAMPLE 13 Purification of the Mouse Anti-human TABM Monoclonal Antibody

[0140] A streptococcal protein-G column (Pharmacia, Sweden) was used toisolate the monoclonal antibody to human TABM. The culture supernatantadjusted to pH7.2 or ascites diluted in PBS (pH 7.2) was filteredthrough a 0.45μ filter and pumped through the column at approximately1.5 ml per min. The column was washed with PBS pH 7.2 until the chartrecorder returned to baseline. The monoclonal antibody was eluted with0.1M glycine-HCl pH2.8 and neutralized with {fraction (1/10)} volume of1M TRIS pH 7.0 followed by dialysis overnight against PBS at 4° C. Theantibody was collected and concentrated in an Amicon (MA. USA) flow cellusing a YM30 filter. The protein content was measured using the Bioradprotein assay micromethod and bovine gamma globulin as the standard.Purified antibody and standard were diluted in PBS.

EXAMPLE 14 Polyacrylamide Gel Electrophoresis in SDS (SDS-PAGE)

[0141] Proteins were mixed 1:1 with SDS-PAGE sample buffer ±5% v/vβ₂-mercaptoethanol and boiled 5 min. The proteins were then resolved in8-25% w/v or 10-15% w/v polyacrylamide gradient gels using the PHAST(Pharmacia) system. Electrophoresed proteins were silver-stained.Molecular weights were determined by the mobilities of pre-stained(Biorad).

EXAMPLE 15 Immunoblotting

[0142] Proteins resolved by SDS-PAGE were electrophoreticallytransferred to a polyvinylpyrollidone (immobilon, Millipore) membrane.The membrane was blocked by incubation in 10 ml 5% w/v powdered milk for2 hr at 37° C. The membrane was washed and then incubated overnight at4° C. with 10 ml of 1:1000 monoclonal anti-TABM antibodies in washbuffer (3). Bound antibodies were detected by the addition of 10 mlalkaline phosphatase-conjugated goat anti-murine IgG (human IgGabsorbed) antibodies and incubation at 4° C. for 2 hrs. The membrane wasthen washed for 1 hr and CSPD chemiluminescent substrate(Boehringer-Mannheim, Indianapolis, Ind.) was added to the membraneaccording to the manufacturer's instructions. Kodak X-ray film wasoverlaid on the membrane and the exposure was held 1 hr, at ambienttemperature, 1 hr, at 37° C. Following exposure the film wasautomatically developed.

EXAMPLE 16 Enzyme Linked Immunosorbent Assay (ELISA)

[0143] Generally, microtiter trays were coated with 100-1,000 ng/wellprotein and blocked with 1% w/v gelatin. A dilution of primary antibody(rabbit, mouse) in wash buffer were incubated 1-1.5 hr at 37° C. andafter washing alkaline phosphatase conjugated goat and rabbit or mouse 1g (whole molecule), antibody was incubated with the microtiter trays for1-1.5 hr at 37° C. Bound antibody was visualized with p-nitrophenylphosphate substrate.

[0144] The screening of fused cells for the production of specificantibody was carried out using the ELISA. A 96-well ELISA plate (Costar,Cambridge, Mass.) was coated with purified human TABM at 500 ng/well in0.06M carbonate buffer, pH 9.5 and incubated overnight at roomtemperature. The plate was washed 5 times with PBS-0.05% v/v Tween-20(PBS-Tw) and blocked with 1% w/w gelatin in carbonate buffer at 37° C.for 90 min. Fifty to 100 μl of culture supernatant from individual wellswith hybrids was added. The samples were incubated at 37° C. for 90 min,followed by washing with PBS-Tw. A peroxidase conjugated sheepanti-mouse immunoglobulins (Silenus, Australia) was diluted {fraction(1/2000)} in PBS-Tw- 1% w/v gelatin and 100 μl placed per wellIncubation was carried out at 37° C. for 90 min, followed by washing.The reaction was developed with 3′,3,5′,5-tetramethylbenzidine (TMB)substrate (KPL, MA) at room temperature until an OD of approximately 2was obtained. The reaction was stopped with 2M H₂SO₄ and the plate CDread at 450 nm.

EXAMPLE 17 Purification of Cohn Fraction III TABM

[0145] Because TABM have the electrophoretic mobility of alpha and(some) beta globulins (2) the inventors tested Cohn Fraction III and IV(gamma globulin fraction) proteins for TABM using the ELISA. CohnFraction III proteins tested positive for TABM, and TABM were notdetected in the gamma globulin fraction.

[0146] To purify TABM, Cohn Fraction III proteins soluble in PBS/ureawere precipitated with 43% (NH₄)₂SO₄, and the precipitated proteins weredissolved in PBS/urea and fractionated by chromatography in sephacrylS-300. Fractions corresponding to molecular weights <30,000-<600,000were obtained, and each fraction was assayed by ELISA for TABM. MostTABM were detected when 50-100 ng of void volume (Fraction I>10⁶ d)(Table 1) proteins were assayed. TABM were also detected when 500 ngsephacryl fraction II proteins were assayed. Immunoglobulins were alsodetected by ELISA in the void volume fraction. Void volume (Fraction I)proteins were reprecipitated with 43% w/v (NH₂)₄SO₄ and solubilised,sephacryl fraction I proteins were absorbed 2× with anti-human Ig,anti-human albumin antibodies conjugated to sepharose beads and thenassayed by ELISA. The absorbed proteins tested position for hTABM, andTcR α chain epitopes, but not β chain epitopes, TGF-β1 and 2 epitopes,but not human immunoglobulin or albumin (FIGS. 1A-C). As shown in FIG.2, reduced Fraction I proteins resolved as Mr 28,000 proteins.Non-reduced proteins did not enter the gel.

EXAMPLE 18 Production of Monoclonal Antibodies

[0147] BALB/c mice were immunized with Cohn Fraction III TABM and seraassayed for anti-TABM antibodies by ELISA against these proteins. Micethat tested positive for anti-TABM antibodies were used as a source ofspleen cells for fusion. Clones producing anti-TABM antibodies weresubcloned. As shown in FIG. 3, culture supernatants from cloneMG3C9-1A12 reacted well with 500 ng purified TABM, but did not react inELISA with 500 ng IgG, IgM, human albumin, IgA, or TGF-β1, 2, 3immunogenic peptides. Other clones (for example, 2B3) did not react withTABM. Clone MG3C9-1A12 was subcloned and assayed for anti-TABM antibody.As shown in FIGS. 4A and B, subcloned, monoclonal MG3C9-1A12 culturesupernatant and ascitic fluid reacted strongly with TABM (FIG. 4A), anddetected less than 50 ng TABM (FIG. 4B). ELISA using heavychain-specific anti-murine Ig demonstrated that MG3C9-1A12 is IgG2b. Inaddition, the monoclonal antibody MG3C9-1A12 reacts with TABM inimmunoglobulin depleted serum (FIG. 4C).

[0148] Serum from a patient with a high titer of TABM to benzoic acidconjugated to HSA (BA-HSA) (toluene is metabolised to benzoic acid) wasadsorbed to sepharose beads conjugated with monoclonal MG3C9-1A12. Thebeads were washed and then eluted with pH 2.8 glycine. Serum moleculesbound by monoclonal MG3C9-1A12 were characterised further by absorbtionto and elution from BA-BSA-sepharose beads. The eluate (benzoic acid-HSAbinding) TABM obtained from the beads were coated to microtiter traysfor ELISA with monoclonal antibodies to TABM or TcR. As shown by ELISA(FIG. 5), monoclonal MG3C9-1A12 bound to BA-HSA binding TABM. Anantibody to Tcr Cα (but not anti-Tcr Cβ or Cδ) also bound toBA-HSA-specific TABM recognised by monoclonal MG3C9-1A12.

EXAMPLE 19 Monoclonal MG3C9-1A12 is Specific for Antigens in Human SerumProteins and extracts of T Lymphocytes

[0149] Because the TABM used as immunogens for monoclonal antibodyMG3C9-1A12 are derived from human serum, the inventors determinedwhether MG3C9-1A12 would bind to unfractionated serum. Dilutions ofhuman serum, fetal bovine and sheep serum, and sera from thecynomologous monkey (Macaca fusicularis) and Rhesus monkeys were coatedto microtiter trays, and as shown in FIG. 6, monoclonal MG3C9-1A12reacted with wells coated with 100 μl of 1:20,000-1:40,000 dilutions.The antibody reacted weakly to cynomologous serum, and more strongly toRhesus monkey serum. However, the “titer” (1:5000) of TABM in Rhesusmonkey serum was not as high as that found for human serum. Themonoclonal antibody (MG3C9-1A12) identified an Mr 28,000 molecularspecies in human serum and the purified TABM used to make the monoclonalantibody (FIG. 7).

[0150] To determine further the specificity of monoclonal MG3C9-1A12,Jurkat (T) and A-1 (B) cells were lysed with Triton X-100, and thelysates mixed with MG3C9-1A12 sepharose beads. After incubation, thebeads were washed and eluted with 0.2M NaCO₃. Aliquots of the eluatewere reduced and resolved by SDS-PAGE. As shown in FIG. 8, Mr 23,000Jurkat cellular proteins were eluted from monoclonal MG3C9-1A12sepharose beads and B-cell proteins were not absorbed to the beads. Inaddition, MG3C9-1A12 did not bind to T or B lymphocytes as judged byimmunofluorescent staining. Furthermore, FIG. 8B shows that B-cells donot contain surface TABM. In contrast, strong binding was shown forT-cells and TABM controls. Examples 20 to 29 relate to the isolation andcharacterization of a TABM specific for the hapten, benzoic acid-humanserum albumin (BA-HSA).

[0151] In a patient with a high titre of TABM to BA-HSA, the inventorspurified TABM to the antigen using a monoclonal antibody to TABM (seeExamples 1-19). The purified TABM comprises a polymer, having monomericunits of molecular weight 28,000. TABM are associated with the cytokineTGF-1. The BA-HSA specific TABM (BA-TABM) also show an apparentcross-reactivity with both dinitrophenol and oxazolone conjugated tohuman serum albumin, and binds weakly to formaldehyde and trimelliticacid (TMA) conjugated to human serum albumin.

[0152] The TGF-β becomes activated when TABM bind to antigen. Althoughthis TABM may suppress cell mediated immunity to specific chemicals,these other properties could result in symptom production.

EXAMPLE 20 Patient

[0153] The serum used to purify TABM specific for BA-HSA was obtainedfrom a 22 year old female patient sensitive to toluene.

[0154] Following toluene exposure (15 ppm for 20 minutes) she reportedfatigue, headache, lower back pain and poor concentration.Neuropsychological assessment demonstrated a decline in performance ontests evaluating immediate and delayed memory recall (Babcock Stores)and psychomotor coordination (Symbol Digit Test) following the tolueneexposure.

[0155] The patient has reported adverse reactions to other chemicals atlow levels of exposure with headache, fatigue, impaired concentration,irritability and muscle pain. Chemicals provoking reactions includedsolvents, perfume and vehicle exhaust.

EXAMPLE 21 Purification of TABM Specific for Benzoic Acid-HSA

[0156] One hundred millilitres of serum were collected from the patientand stored frozen until required. It was thawed, filtered and saturatedammonium sulfate added to 43% w/v. The slurry was then centrifuged at9000 rpm and the supernatant removed. The precipitate was dissolved inPBS pH 7.2 in approximately half the starting volume and dialysedagainst PBS overnight at 4° C. The extract was then passaged through asepharose affinity column (Pharmacia) onto which protein-G purifiedmouse monoclonal antibody to human TABM (MG3C9-1A12) was coupled. Thesample was pumped through the column, following by washing with PBS toremove unbound proteins. The serum total TABM bound by the anti-humanTABM antibody were eluted with pH 2.8 glycine, neutralized with TRIS anddialysed against PBS overnight at 40° C. The purification of antigenspecific TABM to BA-HSA were recovered from a BA-HSA-sepharose affinitycolumn. The antigen is referred to herein as BA-TABM. Before elutionwith pH 2.8 glycine, the column was thoroughly washed with PBS until therecorder returned to baseline. The BA-TABM were then dialysed into 100mM TRIS, 150 mM NaCl pH 7.2 overnight at 4° C. The non-binding proteinswere also collected and dialysed. All samples collected had n-octylglucopyranoside (OG: ICN, CA) added to 30 MM then frozen in aliquots. Aprotein determination was done using the Biorad dye reagent and BSA asstandard.

EXAMPLE 22 Purification of TABM from Cohn Fraction III

[0157] Human serum TABM previously isolated from Cohn Fraction III bygel filtration for the production of the monoclonal antibody (Examples1-19), were used as a positive control in various experiments. Thesewere labelled Cohn FIII-TABM.

EXAMPLE 23 Titration of BA-HSA Specific TABM with Anti-human Antibody(MG3C9-1A12) (FIG. 9)

[0158] Different preparations of human TABM, human IgG (Intragam) forinfusion (CSL, Melbourne), HSA (Calbiochem) and BSA (Sigma) were coatedonto ELISA plates (Costar, Cambridge, Mass.) overnight at roomtemperature. The plate was washed 5 times with PBS-Tween (PT) andblocked with 200 μl 1% w/v HSA (CSL, Melbourne) in PBS for 90 minutes at37° C. One hundred μl protein-G purified mouse monoclonal antibody(MG3C9-1A12) to human TABM (25×supernatant) was diluted {fraction(1/2000)} in PBS-Tw-1% gelatin (PTG), added to the wells and incubatedfor 90 minutes at 37° C. The plate was washed 5 times and peroxidaseconjugated sheep anti-mouse immunoglobulin G (Silenus, Australia)diluted {fraction (1/1000)} in PTG was added to the wells and incubatedfor 90 minutes at 37° C. The reaction was developed with3′,3,5′,5-tetramethylbenzidine (TMB) substrate (KPL, MA) at roomtemperature until an OD of approximately 2 was obtained. The reactionwas stopped with 2M H₂SO₄ and the optical density read at 450 nm.

EXAMPLE 24 The Presence of TABM and IgG in Different TABM Preparationsand Proteins Coated Directly onto ELISA Plates (FIG. 10)

[0159] Different TABM preparations and proteins were serially dilutedand coated directly onto ELISA plates, overnight at room temperature.The presence of TABM were detected using the monoclonal anti-human TABM({fraction (1/2000)}, for 90 minutes at 37° C.) and peroxidase labelledsheep anti-mouse immunoglobulins. {fraction (1/4000)} for 90 minutes at37° C. IgG was detected using a peroxidase labelled sheep anti-human IgG(Silenus, Australia), {fraction (1/4000)} for 90 minutes at 37° C. Thereaction was completed as described above.

EXAMPLE 25 The Presence of Antigen Specific IgG in Different TABM andProtein Preparations (FIG. 11)

[0160] HSA and the haptens BA-HSA, DNP-HSA and FORM-HSA were coated ontoELISA plates at 250 ng/well, in carbonate buffer overnight at 4° C.Patient serum, ‘total’ TABM and BA-TABM were serially diluted and addedto the antigens for 90 minutes at 37° C. After washing, peroxidaselabelled sheep anti-human IgG was applied for 90 minutes at 37° C.

EXAMPLE 26 The Presence of Antigen Specific TABM in Different TABM andProtein Preparations (FIG. 12)

[0161] The hapten BA-HSA and HSA (control) were each plated at 200ng/well. Patient serum. ‘total’ TABM and BA-TABM preparations wereserially diluted and added to the antigens and incubated for 90 minutesat 37° C. The presence of antigen specific TABM were detected using themouse anti-human TABM antibody. Peroxidase labelled sheep anti-mouseimmunoglobulins was then added and incubated for 90 minutes at 37° C.

EXAMPLE 27 Competitive Inhibition ELISA for TABM with a Panel of Haptensor Non-Hapten Molecules Against Plate Bound BA-HSA Antigen (FIG. 13 AND14)

[0162] To determine the antigen specific reactivity of BA-TABM towardBA-HSA and other haptens, a competitive ELISA was used. The ELISA platewas coated with BA-HSA (250 ng/well) in carbonate buffer overnight at 4°C. The plate was washed and blocked with 1% w/v HSA in PBS for 90minutes at 37° C. Serum from the patient with high levels of TABM toBA-HSA was used at a dilution of {fraction (1/400)}, in PBS. Thecompeting antigens or haptens were serially diluted (½) in the dilutedserum to give a range equivalent to 5000-312.5 ng per well. The antigensand haptens tested included human serum albumin (HSA), bovine serumalbumin (BSA). BA-HSA, trinitrophenol conjugated to BSA (TNP-BSA) andformaldehyde conjugated to HSA (F-HSA). The serum and antigen/haptenmixtures were first pre-incubated for 40 minutes at 37° C. before addingto the BA-HSA coated ELISA plate. The plate was then incubated for 60minutes at 37° C. After washing, mouse anti-human TABM was diluted 25({fraction (1/2000)}), added to the plate and incubated for 90 minutesat 37° C., followed by sheep anti-mouse immunoglobulins ({fraction(1/1000)}). The assay was then completed as previously described. Thesame ELISA conditions were used with non-bound organic molecules, exceptthat pre-incubation of competing molecules with BA-TABM and serum wasfor 60 minutes.

EXAMPLE 28 The Serial Dilution of BA-TABM and its Interaction withBA-HSA and other Haptens (FIG. 15)

[0163] A panel of different hapten preparations were diluted incarbonate buffer and plated at 250 ng/well (with respect to HSA) andincubated overnight at 4° C. A serial dilution of the patient serum andthe BA-TABM isolated from this serum were tested against this panel. Theserum and the BA-TABM were serially diluted in PBS: {fraction(1/200)}-{fraction (1/3200)} and 500-31.25 ng/ml, respectively. Onehundred μl were added to wells and incubated at 37° C. for 90 minutes.This was followed by mouse anti-TABM ({fraction (1/2000)}) at 37° C. for90 minutes and peroxidase conjugated sheep anti-mouse immunoglobulins({fraction (1/1000)}) and completed as described above.

EXAMPLE 29 Results

[0164] Neuropsychological Tests

[0165] The results of neuropsychological test are shown in Table 2.

[0166] Protein Determination on BA-TABM

[0167] The purified BA-TABM were collected in a final volume of 10.5 mlfrom a serum volume equivalent to 88 ml. (Partially purified monoclonalantibody bound TABM were left aside for comparative studies). The finalconcentration of BA-TABM was 103 μg/ml. The serum concentration ofBA-TABM was 12.3 μg/ml.

[0168] Detection of TABM in Serum Fractions

[0169] To confirm the presence of TABM at different stages of isolationfrom serum, the TABM were serially diluted and plated. Purified humanIgG was also included as a control. FIG. 9 shows that TABM were detectedusing the monoclonal antibody (MG3C9-1A12) to human TABM as expected. Atypical titration curve was obtained. TABM were present in bothpreparations after affinity isolation and there was no reaction withhuman IgG.

[0170] Detection of IgG and TABM in Samples

[0171] TABM was detected using the mouse anti-human TABM antibody in theBA-HSA, MG3C9-1A12 positive ‘total’ TABM and Cohn fraction IIIpreparations serially diluted and coated directly onto ELISA plates.TABM was not detected in the purified IgG, HSA or BSA samples (FIG. 10).

[0172] An anti-human IgG reacted strongly with purified IgG and not toBSA or HSA. However, there was some binding to the ‘total’ TABMpreparation and perhaps very weak binding to the BA-TABM and Cohnfraction III. This cross reaction could be due to a common epitope onhuman TABM and IgG (FIG. 10).

[0173] To Determine the Presence of Antigen Specific IgG in Serum andDifferent TABM Fractions

[0174] Antigen specific serum IgG binding and serum derived TABMpreparations were tested against different antigens. FIG. 11A shows thatthe patient serum has IgG antibody to the haptens BA-HSA and DNP-HSA. Inthe BA-TABM specific for BA-HSA there was no antigen specific IgGdetected (FIG. 11C). In the MG3C9-1A12 positive ‘total’ TABM, there maybe a little IgG present to BA-HSA and DNP-HSA (FIG. 11B). However, thiscould be due to cross-reaction by the polyclonal anti-human IgG antibodywith TABM molecules.

[0175] Titration of BA-TABM Against BA-HSA

[0176] A typical titration curve was obtained for the interaction ofserially diluted BA-TABM against the hapten BA-HSA. There was noreaction with the HSA control. Concentrations of specific TABM of lessthan 100 ng/mL were detectable in the assay (FIG. 12). A similar curvefor the MG3C9-1A12 positive ‘total’ TABM eluted from the mouseanti-human TABM affinity column was obtained. TABM specific to BA-HSAwere also detected in the patient serum at dilution of {fraction(1/3200)}.

[0177] Inhibition Studies on Binding of BA-TABM to BA-HSA

[0178] The pre-incubation of BA-TABM to plate bound BA-HSA or DNP-HSAinhibited the binding of BA-TABM to plate bound BA-HSA in a dosedependent manner. A typical titration curve was obtained (FIG. 13).Weaker inhibition was also observed with pre-incubation of BA-TABM withForm-HSA and TMA-BSA.

[0179] Inhibition Studies on Binding of BA-TABM to BA-HSA and FreeChemicals

[0180] BA-TABM was pre-incubated with the hapten BA-HSA and the unboundmolecules benzoic acid, para-aminobenzoic acid and toluene to seewhether TABM recognize small chemical molecules unbound to protein.

[0181] There was inhibition of the BA-TABM binding to plate bound BA-HSAby the competing BA-HSA (FIG. 14). There was no inhibition of BA-TABM bythe free molecules. Similar results were obtained when BA-TABM waspre-incubated with DNP-lysine and o-cresol.

[0182] Reaction of BA-TABM with a Panel of Haptens

[0183] Not only did BA-TABM bind strongly to BA-HSA, but also to DNP-HSAand OX-HSA with comparable titres. There was also weaker binding toForm-HSA and TMA-HSA (FIG. 15). Serum TABM also showed strong binding toDNP-HSA and OX-HSA.

[0184] Examples 30 to 38 relate to the clinical and immunologicalresponses in subjects sensitive to solvents.

[0185] To evaluate the role of immunological responses in chemicalsensitivity, the inventors measured serum IgG and TABM levels specificfor an antigen produced by conjugating benzoic acid (a major metaboliteof toluene) to human serum albumin (BA-HSA). These measurements wereperformed in a group of patients clinically documented to be sensitiveto toluene and a control group. The results indicate an associationbetween an elevation in serum TABM specific for BA-HSA and symptoms ofchemical sensitivity and suggest that measurement of TABM againsthaptens may be of value in assessing patients sensitive to chemicals.

EXAMPLE 30 Patients

[0186] Twenty toluene sensitive patients were studied (elevan female,nine male). Their average age was 39.5 years). Some patients had ahistory of adverse reactions to chemicals, including solvents. Otherswere only subsequently shown to be sensitive to chemicals. Presentingsymptoms including fatigue, irritability, headache, musculoskeletal painand poor concentration. Additional symptoms were soreness of the throat,sleep disturbance, post-nasal drip and nausea. More than half thepatients had a history of occupational exposure to petroleum basedchemicals, particularly to solvents (Table 3).

[0187] Patients were considered to be sensitive to the solvent tolueneon the basis of developing symptoms and cognitive impairment following acontrolled exposure. Using a specially designed testing booth, they werechallenged with toluene at a concentration of 15 parts per million for20 minutes. Symptoms before and subsequent to the chemical exposure wererecorded.

[0188] Neurobehavioural tests were used to evaluate congnitivefunctioning pre and post challenge. The tests chosen were similar tothose previously shown to be effective in documenting symptomsassociated with solvent exposure (4). Focal length was an additionalmeasurement included in the battery to assess the often cited subjectiveresponse by patients of blurred vision.

[0189] The battery provided estimates of immediate and delayed verbalmemory recall (Babcock stories (5)), information processing times(Simple Reaction Time), concentration and attention (Letter Cancellationadapted from Lezak (5), and STROOP Colour Word (6)), psychomotorcorrdination (Symbol Digit (7)) and visual acuity (Focal Length). Theresults of pre versus post challenge data were analysed using theWilcoxon matched pairs signed ranks test.

[0190] For purposes of analysing data, the patients were classified intwo groups, according to the duration of chemical exposure:

[0191] Group 1 (7 patients)—exposure range 14 to 25 years;

[0192] Group 2 (10 patients)—exposure range up to 13 years;

[0193] Only patients with substantial chemical exposure were considered.

[0194] A second classification was made in terms of the type of chemicalexposure incurred: Group A (6 patients) were principally exposed toheavy metals and solvents. Group B (8 patients) were exposed to othermiscellaneous chemicals including hair dyes and pesticides.

[0195] Evaluation of chemical exposure was based on: 1. Work historysince leaving school; 2. Accommodation history, including proximity toindustry airports, petrochemical plants, major traffic routes, crops andorchards and frequency of pest control spraying; 3. Hobbies and otheractivities: frequency of home renovation, furniture restoration, cardetailing and repair; cottage industries were considered under thisheading; 4. The occupation and hobbies of other family members wereassessed as potential sources of exposure to chemicals.

[0196] The intensity and duration of exposure and type of chemical wereconsidered in the assessment of exposure to chemicals.

EXAMPLE 31 Controls

[0197] Sixteen control subjects were studied (eight females and eightmales). The average age was 32 years. Control subjects had no history ofadverse reactions to chemicals and were reportedly in good health. Theywere not troubled by persistent symptoms such as fatigue, headache,musculoskeletal pain or poor concentration. The had no significanthistory of solvent exposure.

EXAMPLE 32 Serum Samples

[0198] Blood was collected by venipuncture, allowed to clot andcentrifuged to collect the serum. Serum was stored frozen in multiplealiquots.

EXAMPLE 33 Anti-human TABM Antiserum (R28)

[0199] The R28 antiserum is a polyclonal rabbit antiserum directedagainst human T-cell derived antigen specific binding molecules (TABM).It was prepared by isolating TABM from human serum by ammonium sulphateprecipitation and ion exchange chromatography (8). Any contaminatingimmunoglobulin and albumin were removed by passage through an anti-humanimmunoglobulin and an anti-human albumin affinity column. The rabbitantiserum that was raised to TABM was also passed through a humanimmunoglobulin and albumin affinity columns to remove antibodies thatcould react with human IgG and albumin. There is no cross reactivity ofR28 antibody with human IgG or IgM or albumin as tested by ELISA.

EXAMPLE 34 Antigen

[0200] In these experiments, the inventors used para-amino benzoic acidthat was conjugated to human serum albumin (BA-HSA) as antigen(Immunosciences Lab Inc, Los Angeles) and was made by the diazotizationmethod (9).

EXAMPLE 35 ELISA Assay

[0201] The BA-HSA was diluted in 0.06M carbonate buffer, pH 9.6 and 100microliters (500 ng) dispensed into wells of NUNC Maxisorp ELISA plates(Nunc, Denmark). The plates were incubated overnight at 4° C. Followingfive washed with PBS-0.05% v/v Tween-20 (PBS-Tw), the plates wereblocked with 200 μl of 1% w/v gelatin in carbonate buffer for 75 min at37° C. After 5 washes, 100 μl of serum diluted {fraction (1/500)} inPBS-Tw 1% w/v gelatin for determination of IgG or {fraction (1/20)} forTABM was added to the wells and incubated for 90 min at 37° C. Afterwashing the plates, 100 μl of {fraction (1/3000)} sheep anti-humanIgG-peroxidase (Silenus Laboratories, Australia) diluted in PBS-Tw 1%w/v gelatin was added per well and incubated for 90 min at 37° C. Thecolour reaction was generated by adding 100 μl of3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, Gaithersberg, Md.)per well and incubating at room temperature for the colour to develop.The reaction w%as stopped with 100 μl of 2M H₂SO₄. For TABM, rabbitanti-human TABM (R28 serum) was diluted {fraction (1/300)}, added co thewells and incubated for 90 min at 37° C. This was followed by 100 μl of{fraction (1/500)} sheep anti-rabbit IgG peroxidase (SilenusLaboratories, Australia). After 90 min at 37° C. the reaction wasdeveloped and stopped as above. Control antigens included gelatin andHSA plated at 500 ng/well. All incubated were carried out in ahumidified box. Plates were then read at 450 nm. All samples were testedin duplicate.

EXAMPLE 36 Standards

[0202] The ELISA assays were first run to determine optimumconcentrations of all reagents, followed by evaluation of some serumsamples. Serum samples with high activity (OD) were pooled (equalvolumes), aliquoted and frozen. For each ELISA assay a fresh ampoule wasserially diluted and used as an assay standard. Arbitrary units wereassigned to each standard serum pool as follows: IgG to BA-HSA 100,000U/ml; and TABM to BA-HSA 10,000 U/mi. Serial (½) dilutions of pooledstandard serum were: IgG, {fraction (1/200)}-{fraction (1/3200)}; andTABM, {fraction (1/20)}-{fraction (1/640)}. The units of activity ofeach serum sample were determined after plotting standard curves usingthe Beckman Immunofit EIA/RIA analysis program (v 3.0).

EXAMPLE 37 Competitive Inhibition ELISA for Antigen Specificity

[0203] To determine the antigen specificity of a serum toward the BA-HSAhapten, a competitive ELISA was used. The ELISA plate was coated withBA-HSA (250 ng/well) in carbonate buffer overnight at 4° C. A serum froma patient with high levels of (R28+) TABM to BA-HSA was used at adilution of {fraction (1/200)}. The competing antigens or haptens wereserially diluted (½) in the serum to give a range equivalent to5000-312.5 per well. The antigens and haptens tested included humanserum albumin (HSA), bovine serum albumin (BSA), BA-HSA, trinitrophenolconjugated to BSA (TNP-BSA) and formaldehyde conjugated to HSA (F-HSA).The serum and antigen/hapten mixtures were first preincubated for 30 minat 37° C. before adding to the BA-HSA coated ELISA plate. The plate wasthen incubated for 60 min at 37° C. After washing R28 was diluted({fraction (1/400)}), added to the plate and incubated for 90 min at 37°C. The assay was then completed as previously described.

EXAMPLE 38 Results

[0204] Clinical Observations

[0205] Details on the patient group are listed in Table 3. Disabilitywas clinically rated as mild, moderate or severe depending on severityof symptoms and interference with lifestyle, including whether employedor not. Severely affected patients were unable to work and thosemoderately affected required a major change in their work environment toremain employed. The severity of the reaction to toluene was rated 0 to+++. Typical symptoms produced on toluene challenge included fatigue,poor concentration, headache and muscle pains.

[0206] Neuropsychological Testing

[0207] The results of neuropyschological testing of patients are shownin Table 4. The test subjects showed a significant deterioration inperformance post challenge on immediate and delayed memory tests (FIG.16 and 17) and, less notably, on digit-symbol (FIG. 18) and lettercancellation error tests (FIG. 19).

[0208] IgG Assay

[0209] There was no significant difference (p=0.535) in serum levels ofantigen specific IgG to the hapten-HSA antigen (BA-HSA) between controlsand patients (FIG. 20). However, in both groups a number of individualsshowed low levels of IgG to the antigen.

[0210] TABM Assay

[0211] The patient group showed higher levels of TABM to the BA-HSAantigen than controls (FIG. 21), p=0.002. Out of a total of 36 assays onpatients and controls, the 12 highest values for TABM were all from thepatient group. As mentioned, the R28 antibody detects human TABM thatbind antigen specifically.

[0212] The specificity of the TABM to BA-HSA was also tested. A serumpositive for TABM specific to BA-HSA did not show binding to HSA, BSA,F-HSA, TNP-BSA, or the cows milk protein β-lactoglobulin as detectedusing the R28 antiserum. In a competitive binding ELISA, pre-incubationof the patient serum with BA-HSA inhibit binding to BA-HSA bound to theplate wells. The inibition occurred in a dose dependent manner (FIG.24). No inhibition of serum TABM binding to BA-HSA on the well wasobserved when the serum was pre-incubated with HSA, BSA, F-HSA orTNP-HSA. In these experiments the R28 antiserum was also treated toremove any rabbit IgG to BA-HSA by passage through a BA-HSA affinitycolumn.

[0213] Association Between TABM Level and Clinical Severity

[0214] The TABM level correlated with clinical assessment of disability,p=0.008.

[0215] Comparison of TABM Score and Severity of Reaction to Toluene

[0216] There was no correlation between the severity of symptomsproduced by toluene exposure and the TABM score, p=613.

[0217] Comparison of TABM Level and Changes in Performance onNeuropsychological Tests

[0218] Statistical analysis showed significant correlations between TABMand changes in both focal length and in STROOP (colour word) postchallenge (Table 5 and FIG. 22). There was no correlation between TABMlevels and other neuropsychological tests.

[0219] Comparison of TABM Levels and Chemical Exposure

[0220] TABM levels were higher in subjects with a longer history ofchemical exposure (Group 1) p=0.0125 (FIG. 23). No other differences inTABM levels were evident in comparisons between the groups.

[0221] Comparison of Neuropsychological Tests with Groupings Related toChemical Exposure

[0222] Subjects with a longer history of exposure (Group 1) showed agreater deterioration of the STROOP (Colour Word) Test (p=0.017)indicating greater attention difficulties. Subjects with a shorterhistory of exposure (Group 2) showed a greater deterioration inperformance on the Prose Memory (delayed) Score (p=0.017) and the DigitSymbol Test (p=0.039). The latter test assesses psychomotorco-ordination (Table 6). Subjects with a history of exposure to heavymetals and/or solvents (Group A) had a significantly lower Prose Memory(immediate) Score (p=0215) and shorter reaction time (p=0.0441 ) priorto their challenge, as compared to Group B (Table 7).

SUMMARY

[0223] Twenty patients were demonstrated to be sensitive to the solventtoluene when exposed to thus chemical at a concentration of 15 parts permillion for 20 minutes. Neuropsychological tests on patients assessedbefore and after toluene exposure showed impairment in cognitivefunctioning, with a deterioration in short and long term memory andpsychomotor cordiniation. Total IgG and T-cell antigen binding molecules(TABM) were measured against an antigen prepared by conjugatingpara-amino benzoic acid to human serum albumin (BA-HSA) in the patientsand 16 controls. There was no significant difference in the IgG levelsto the antigen, but the levels of TABM against the BA-HSA weresignificantly elevated in those subjects sensitive to toluene. There wasno significant associations between the TABM levels and, a) poorerperformance on the STROOP (colour word) test, b) shift in focal lengthafter toluene exposure, c) clinical assessment of disability and d)longer history of chemical exposure. The measurement of TABM againstchemical haptens may be of value in assessing patients sensitive tochemicals.

[0224] Examples 39 to 47 relate to the measurement of TABM and IgG tomannan from Candida albicans in patients with recurrent thrush.

[0225] Numerous studies have indicated the importance of cell mediatedimmunity (Th1 response) in protection against Candida albicansinfection, particularly at mucosal surfaces. This type of response alsoappears important in the prevention of vulvo-vaginal candidiasis. Inpatients susceptible to this type of Candida infection, studies indicatea local impairement of cell-mediated immunity to Candida albicans, eventhough the Th1 immune response to the yeast appears systemically intact.

[0226]Candida albicans is associated with numerous antigens. Certainantigens appear important in stimulating protective cell mediatedimmunity. Other antigens, particularly the carbohydrate mannan, arethought to be implicated in immune suppression.

[0227] The monoclonal antibody (MG3C9-1A12) described in Examples 1-19is specific for human TABM. This antibody does not react withimmunoglobulin G or albumin. An ELISA assay has been developed using themonoclonal antibody to measure TABM specific to whole Candida extractand to mannan prepared from Candida albicans using the Peat and CTAB(cetryltrimethylammonium bromide) methods. Serum levels of antigenspecific TABM (and IgG) were measured in patients susceptible tovulvo-vaginal candidiasis and a control group.

EXAMPLE 39 Subjects

[0228] Women attending the Sexual Health Centre, Melbourne. Victoria anda family planning clinic were included in the study.

[0229] Controls had not had thrush for one or more years and thrush wasnot an ongoing problem.

EXAMPLE 40 Serum Samples

[0230] Institutional ethics approval was obtained for the studs.Informed consent from the blood donors was obtained.

[0231] Ten millilitres of blood were collected by venipuncture intovacutainer tubes. The blood was allowed to clot at room temperature andcentrifuged to recover the serum. The serum was frozen at −20° C. inmultiple aliquots of 0.5-1 mL. A fresh aliquot of serum was used foreach assay.

EXAMPLE 41 Candida Antigens

[0232] Two different preparations of mannan derived from C. albicanswere used. The cetryltrimethylammonium bromide (CTAB) method involvescomplexing with the mannan which is subsequently isolated. Preparationof mannan (PEAT) involves degrading under alkaline conditions andprecipitation with Fehling's solution. The mannans are a highly branchedglycoprotein containing essentially mannose and less than 10% of themolecular weight is protein.

EXAMPLE 42 Monoclonal Antibody to Human TABM

[0233] The mouse monoclonal antibody (MG3C9-1A12) specificallyrecognises human TABM (see Examples 1-19). The monoclonal antibodypreparation used in this study was purified from culture supernatant (25fold) using Protein-G sepharose (Pharmacia) and is stored in PBS withsodium azide at 4° C.

EXAMPLE 43 ELISA Assays

[0234] Extensive ELISA experiments were carried out to determine theoptimum conditions for the measurement of TABM and IgG levels to each ofthe Candida antigens.

EXAMPLE 44 Serum Standard for ELISA Assays

[0235] Equal volumes of serum from six patients with recurrent thrushwere pooled, aliquoted in 0.2 ml volumes and stored frozen untilrequired. The serum pool was serially diluted by ½ in PBS-Tw-1% w/wgelatin (PTG) for IgG and in PBS for TABM. A standard curve wasgenerated after plotting optical density (OD) against arbitrary‘Units/ml’ with respect to the dilution made. The dilution range of theserum standard and the number of arbitrary units assigned to theundiluted serum for each ELISA assay is as follows: TABM to CTAB andPEAT mannan, {fraction (1/20)}-{fraction (1/320)} and 1,000 units/ml.TABM to Hollister-Stier skin test antigen, {fraction (1/25)}-{fraction(1/3,200)} and 5.000 Units/ml: IgG to CTAB and PEAT mannans, {fraction(1/500)}-{fraction (1/128,000)} and 10⁶ Units/ml: and IgG toHollister-Stier skin test antigen, {fraction (1/500)}-{fraction(1/64,000)} and 10⁶ Units/mL. All dilutions of the standard and sampleswere tested in duplicate. Each dilution was assigned the appropriatenumber of ‘units/ml’. The units of activity of each serum sample weredetermined after plotting standard curves using the Beckman ImmunofitEIA/RIA analysis program (v 3.0).

EXAMPLE 45 ELISA for Detection of Human Immunoglobulin G Specific toCandida albicans CTAB and PEAT Mannans

[0236] The mannan preparations were diluted in PBS pH 7.4 and coatedovernight at 37° C. on Falcon (Becton Dickinson, N.J.) ‘pro-bind’plates. After washing with PBS-0.05% v/v,Tween 20, (PBS-T), the plateswere blocked with 1% w/v gelatin in carbonate buffer pH 9.6 for 90 minat 37° C. and rewashed. The standard serum pool was serially diluted asabove. Serum samples were diluted {fraction (1/5000)} in PTG and 100 uladded per well in duplicate and incubated for 90 min at 37° C. Theplates were washed and 100 μl affinity purified peroxidase conjugatedsheep anti-human immunoglobulins (Silenus, Australia) was added({fraction (1/10,000)}) and incubated for 90 min at 37° C. Followingwashing, 100 μl 3,3′,5,5′-tetramethylbenzidine hydrochloride (TMB)substrate (KPL, Gaithersburg, Md.) was added and the reaction allowed toprocede at room temperature until an optical density (O.D.) ofapproximately 2 was obtained. The reaction was stopped with 2M H₂SO₄ andthe plates read at 450 nm.

EXAMPLE 46 ELISA for Detection of Human Immunoglobin G Specific toHollister-Stier Candida albicans Skin Test Antigen

[0237] The antigen preparation was diluted in 0.06M carbonate buffer pH9.6 and coated overnight at 4° C. on Costar (Cambridge, Mass.) E.I.A.plates. After washing with PBS-T, the plates were blocked with 1% w/vgelatin in carbonate buffer for 90 min at 37° C. and rewashed. The serumsamples were diluted {fraction (1/2000)} in PTG and 100 μl plated perwell in duplicate and incubated for 90 min at 37° C. The plates werewashed and 100 μl affinity purified peroxidase conjugated sheepanti-human immunoglobulins ({fraction (1/5,000)} in PTG) was added andincubated for 90 min at 37° C. Following washing, substrate was addedand the reaction completed as above.

EXAMPLE 47 ELISA for Detection of Human TABM Specific to Candidaalbicans CTAB AND PEAT Mannans

[0238]Candida albicans mannans were bound to Falcon ELISA plates asabove. The plates were blocked with 1% w/v human serum albumin (HSA) inPBS and washed. Serum samples were tested at {fraction (1/50)} dilutionin PBS and 100 μl added per well and incubated. Protein-G purified mousemonoclonal anti-human TABM antibody was diluted {fraction (1/1,000)} inPTG, and 100 μl added to the wells, incubated and washed. One hundred μlof affinity purified sheep anti-mouse immunoglobulins (Silenus,Australia) diluted {fraction (1/500)} in PTG was added and incubated.The reaction was completed as above.

EXAMPLE 48 ELISA for Detection of Human TABM Specific forHollister-Stier Candida Skin Test Antigen

[0239] The antigen preparation was diluted in 0.06M carbonate buffer pH9.6 and coated overnight at 4° C. on Costar E.I.A. plates. After washingwith PBS-T, plates were blocked with 1% w/v gelatin in carbonate. Serumsamples were tested at {fraction (1/200)} dilution in PBS. Protein-Gpurified mouse monoclonal anti-human TABM antibody was diluted {fraction(1/1,000)} in PTG, added to the wells, incubated and washed. An affinitypurified sheep anti-mouse immunoglobulins was diluted {fraction (1/500)}in PTG. The reaction was completed as above.

EXAMPLE 49 Results

[0240] Immunoglobulin G Levels to Candida albicans Antigens

[0241] A comparison of IgG levels between patients and controls to C.albicans CTAB and PEAT mannans showed a significant difference (P=0.048and 0.032, respectively). There was no significant difference in IGlevels between patients and controls to the Hollister-Stier skin testpreparation (P=0.079 1).

[0242] TABM Levels to Candida albicans Antigens

[0243] TABM levels between patients and controls were significantlydifferent to the CTAB mannan preparation (P=0.0291). There was nosignificant difference in levels to the PEAT mannan (P=0.0721) nor tothe Hollister-Stier preparation (P=0.2166) between patients andcontrols.

[0244] Examples 50 to 57 relate to the action of TABM on sensory nerveendings.

[0245] It has been proposed that chemical sensitivity is due to theaction of specific chemicals on afferent nerves. This could produceneurogenic information in tissues exposed to chemicals. The centralnervous system and other tissues could also be affected by the relay ofneural signals to the central nervous system and the antidromicpropagation along other sensory nerves. The latter process is describedas neurogenic switching.

[0246] The difficulty with this theory is that although many chemicalsmay stimulate afferent nerves by acting on irritant receptors, anadditional action on nerve endings is required to explain the effectsreported by chemically sensitive patients. Tolerance normally developsto the effects of chemical irritants. It has been further proposed thatin chemically sensitive patients, there is mucosal injury, allowinggreater access of chemicals to sensory nerve endings. Where chemicalsensitivity is reported after a high chemical exposure, it is suggestedthat the mucosal barrier is disrupted. Although such an event perhapscauses adverse reactions to chemicals during the period immediatelyfollowing a high chemical exposure, it seems unlikely to account for thelong term problems experienced by chemically sensitive patients.

[0247] Immune sensitisation to chemicals is another possible consequenceof chemical exposure. This process could result in the local release ofinflammatory mediators and cytokines. Cytokines may act on sensory nerveendings with propagation of neural signals to the central nervoussystem. In the case of interleukin-1, the hypothalamus and centresinvolving the regulation of mood and pain are affected by this process.

[0248] In the following Examples, the inventors show that the cytokineTGF-β is associated with TABM purified from the serum of a patientsensitive to the solvent toluene. These TABM are specific to benzoicacid conjugated to human serum albumin (BA-HSA), benzoic acid being animportant metabolite of toluene. It is further demonstrated that bothTGF-β and the TABM enhance the release of neuropeptides from sensorynerves in a dose dependent manner and in both cases the effect isblocked by anti-TGF-β antibody. When the antigen BA-HSA is added, theeffect of TABM may be enhanced or reduced, depending on the ratiobetween TABM and antigen.

[0249] This study may explain:

[0250] (a) how neurogenic inflammation could be produced in chemicallysensitive patients with possible effects on the central nervous system;and

[0251] (b) how the effects on sensors nerves may vary with chemicalconcentration, but not in a dose dependent manner.

[0252] A vacuum-induced blister model in the footpad of anaesthetisedrats is used to induce an inflammatory response in naive skin by (a)electrical stimulation (ES) of the distal end of the cut sciatic nerveat 20 v, 5 Hz. 2 ms for 1 min or (b) superfusion of the sensoryneuropeptide; substance P (SP) over the blister base.

EXAMPLE 50 Animals

[0253] Male outbred Sprague-Dawley rats with an average weight of250-300 gm were used (n=6-8 per group). Anaesthesia was induced withpentobarbitone sodium (Nembutal, 60 mg/kg, i.p.). To ensure that ratsremained under a constant stage of surgical anaesthesia additional doses( 15 mg/kg) were administered during the experiment. The absence of aneyelash reflex was used as an indication of the level of unconciousness.At the end of the experiment, animals were killed by barbituate overdose(100 mg/kg).

EXAMPLE 51 Blister Induction and Experimental Set Up

[0254] A blister was induced on the hind footpad on the anaesthetisedrat by applying a vacuum pressure of −40 kPa to the glabrous skin forapproximately 30 mins, using a metal suction cap heated to 40° C. by anattached heating element.

[0255] When a blister was established, the surface epithelium wasremoved and a perspex chamber with inlet and outlet ports was fixed overthe blister base. Perfusion of the drugs over the blister was maintainedat 4 ml/h by a peristaltic pump (Microperpex S, LKB, Sweden). Bothperfusion and body temperature were kept at 37° C.

[0256] An initial 15 min equilibration with Ringer's solution (NaCl 9.4g, KCl 0.42 g, CaCl₂ 0.48 g, NaHCO₁ 0.2 g in 1000 ml dH₂O) was allowedbefore each experiment, during which time a stable baseline wasestablished.

EXAMPLE 52 Antidromic Electrical Stimulation (FOR TGF-β)

[0257] The right sciatic nerve in the mid-thigh region of theanaesthetised rat was dissected free and cut as proximally as possible.The distal portion of the cut nerve was placed over bipolar platinumelectrodes and immersed in liquid paraffin warmed to 37° C. andcontained in a bath that was formed by raising the skin flaps of thewound. The distal end of the sciatic nerve was stimulated with a GrassS48 stimulator at 20V, 5 Hz, 0.5 ms square waves for 1 min. Theseparameters were previously shown to stimulate sensory nerves withsubsequent release of sensory peptides.

EXAMPLE 53 Peptide Perfusion

[0258] The experimental protocol consisted of a pre-stimulation periodwith Ringer's solution (two 10 min periods), followed by a 10 minperfusion of sodium nitroprusside (SNP) at 100 μM. The latter is adirect smooth muscle vasodilator that is used in the experiment tocontrol for the variability in smooth muscle reactivity between rats.This was followed by two 10 min periods with Ringer's solution tore-stabilise the baseline. A three 10 min stimulation periods witheither SP (1 μM) alone or in the presence of TABM (0.1 or 1.0 μg/ml).

[0259] Finally, there was a 10 min post-stimulation period withperfusion of Ringer's solution. TABM were perfused over the blister baseduring the second 10 min of re-establisation periods as well as duringthe three stimulation periods. In some experiments, TABM were perfusedat 0.1 μg/ml for 20 min.

[0260] In experiments where the effect of antigen was tested, antigen(0.001 μg) was perfused for 10 min prior to and together with TABM andSP.

EXAMPLE 54 Measurement of Local Blood Flow

[0261] For measurement of local blood flow, a probe attached to a lasterDoppler flowmeter (Periflux, PF2B, Perimed, Sweden) was insertedvertically through a third port in the perspex chamber above the blisterbase. Relative blood flow was recorded over time on a chart recorder andmeasured as the surface area under the response curve in cm² using adigital planimeter.

EXAMPLE 55 Materials

[0262] Substance P (Auspep, Australia) were dissolved in Ringer'ssolution. All other common chemicals and solvents were of analyticalgrade from various commercial sources.

EXAMPLE 56 Expression of Data and Statistical Analysis

[0263] Vasodilator responses were measured as the area under theresponse curve (cm²) using a digital planimeter (Tamaya, Japan). Forvascular responses to ES the area was measured for 20 min followingstimulation. For SP response which is characterised by gradualtachyphylaxis, this was the area during the 30 min perfusion. For SNPresponses that are characterised by maintained responses, this was thearea under the curve during 10 min of perfusion. Results are expressedas mean ±SEM. Statistical analyses were perfomed using one way analysisof variance (ANOVA) with a priori planned contrasts. SNP responses wereused as a covariate. Type 1 error was set at α=0.05 and p value of 0.05was considered significant.

EXAMPLE 57 Results

[0264] The Vascular Response to ES in Capsaicin Pretreated Rats

[0265] Neonatal capsaicin pretreatment was conducted when rats were 3-4days old. A single subcutaneous injection of 50 mg/kg capsaicin causesselective destruction of 80% of sensory C-fibres, these bring termedcapsaicin-pretreated rats. These rats are utilised to investigate therole of sensory nerves in different physiological responses. When thevascular response to ES was examined in capsaicin-pretreated rats, theresponse profile was characterised by quicker tachyphylaxis compared tocontrol and the area of the response was only 11.7±1.1 cm².

[0266] Effect of TGF-β on the Vascular Response to Antidromic ES of theSciatic Nerve

[0267] Electrical stimulation of the cut/exposed sciatic nerve resultedin an increase in local blood flow in base of blisters induced in naiveskin. The increase was maintained for a period of over 20 minutes. Thearea under the response curve for 20 minutes was 18.1±0.9 cm². TGF-β (10pg/ml) perfused over the blister base for 10 min prior to ES as well asduring the post stimulation period, did not alter the baseline butenhanced the response from 18.1±0.9 cm² to 31.44±3.2 cm².

[0268] Effect of TGF-β on the Vascular Response to ES inCapsaicin-pretreated Rats

[0269] When TGF-β was used to 10 pg/ml it did not alter the baseline inthese rats and it also had no effect on a subsequent response to ES. Theresponse to ES in capsaicin-pretreated rats in presence of TGF-β was12.1±1.0 compared to a ES response in the absence of TGF-β 11.7 ±1.1cm². The fact that TGF-β at the concentration of 10 pg/ml did not alterthe response to ES in capsaicin-pretreated rats while it significantlyenhanced the response in control rats suggests that the response toTGF-β at this concentration is mediated via an action on sensory nerves.

[0270] Effect of TABM on the Vascular Response to SP, Enhancement byAntigen

[0271] Perfusion of SP (1 μM), alone over the base of a blister inducedin naive skin resulted in a vasodilatation response that reached itsmaximum within 3-5 min of initiation of perfusion. The responsegradually diminished and reached the basal level in 15-20 min despitecontinuous perfusion of SP for 30 min. Perfusion of TABM (0.1 & 1.0μg/ml) over the blister base prior to and together with SP (1 μM)significantly enhanced the vasodilatation response to SP in a dosedependent manner from 44.1±3.9 cm² to 63.4±4.8 cm² & 84.5±4.6 cm²,respectively. TABM alone had no effect on basal blood flow.

[0272] Effect of Duration of Exposure to TABM on the Vascular Responseto SP

[0273] In another experiment TABM was perfused at 0.1 μg/ml either for10 or 20 min over the blister base prior to and then together with SP (1μM). Perfusion of TABM for 10 min enhanced the response to SP from44.1±3.9 cm² to 63.4±4.8 cm². On the other hand, prolonged exposure ofthe blister base to TABM (20 min) greatly enhanced the response to90.6±5.2 cm². This degree of enhancement is equivalent to that inducedby the higher dose (1.0 μg/ml) which increased the response to 84.5±4.6cm².

[0274] Effect of Antigen Pre-exposure on the Effect of TABM

[0275] Pretreatment with antigen (0.001 μg/ml for 20 min) on theinflammatory response to SP. Anti-TGF-β antibody reduced the responsefrom 90.6±5.2 cm² to 49.5±3.8 cm².

[0276] Effect of Anti-TGFβ Antibody on the Effect of TABM on theInflammatory Response to SP

[0277] Pretreatment with anti-TGFβ antibody (0.1 μg/ml) significantlyinhibited the enhancing effect of TABM (0.1 μg/ml for 20 min) on theinflammatory response to SP. Anti-TGFβ antibody reduced the responsefrom 90.6±5.2 cm² to 49.5±3.8 cm².

[0278] Examples 58 to 71 relate to the production of serumimmunoglobulins and TABM specific for cow's milk antigens in adultsintolerant to cow's milk.

[0279] While IgE-mediated reactions to foods are reasonably wellunderstood, the role of adverse reactions to foods in clinical problemssuch as migraine, irritable bowel syndrome and the chronic fatiguesyndrome remains controversial. Although a number of studies havereported the importance of diet in these disorders (10-16), otherstudies are in disagreement (17,18). These differing results may be dueto methodological variations in the studies, and to difficulties insymptom assessment.

[0280] There have been a number of explanations advanced to explaindelayed reactions to foods including pharmacological, psychosomatic andimmunological reasons.

[0281] Although IgE antibody levels in various food stuffs have beendetermined in a number of disorders such as migraine (12), themeasurement of IgG antibodies has been limited. Relatively little workhas been done in measuring total IgG and IgG subclasses against commonfoods in adults (9). Also, there are difficulties in the interpretationof such data as it is unclear whether the production of IG antibodies tofoods is a physiological response.

[0282] There is little information available concerning the role of a Tlymphocyte response to food antigens.

[0283] In the following Examples, the inventors measured serum IgG, IgGsubclasses (IgG1, IgG2, IgG3, IgG4), IgE and TABM specific for threeprincipal antigenic milk proteins; β-lactoglobulin, α-lactalbumin andcasein in milk-intolerant patients and controls. TABM-specific forβ-lactoglobulin (BL-TABM) in a milk-intolerant patient's serum werepurified by affinity for β-lactoglobulin were found to be Mr 28,000,46,000 polypeptides (after reduced) that are not recognized byantibodies to human immunologlobulin chains. Moreover, the non-denaturedantigen affinity-purified TABM are associated with TGF-β1 and TGF-β2.

EXAMPLE 58 Patients

[0284] Fifteen milk-intolerant patients were studied (13 female, 2male). The average age was 39.5 years. Patients had presented with arange of symptoms including abdominal pain, diarrhea, headache,musculoskeletal pain and fatigue. Two subjects had severe migraine. Milkintolerance was diagnosed on the basis of the development of symptomsafter open challenge with half a litre of milk following symptomimprovement on elimination die. Patients were observed for up to 8 hoursafter the challenge and symptoms recorded for two days. Symptomsreported following the challenge and symptoms included headache, fatigueand musculoskeletal pain (Table 8). Patients had not ingested milkproducts for at least two weeks both prior to the milk challenge andwhen blood samples were taken.

[0285] The patient from whom blood was obtained to purify TABM wasclinically intolerant to cow's milk. The ingestion of 250 ml of cow'smilk produced numerous symptoms over the following 24-48 hours. Symptomswere noted within 1-2 hours of milk ingestion and included abdominaldistension, abdominal pain, diarrhea, fatigue and agitation. Difficultyin concentrating, mental unrest and sleep disturbance were also observedto occur. A skin prick test for cow's milk was negative and a lactosetolerance test was normal. The patient had presented with fatigue, sleepdisturbance, headache, poor concentration and postnasal drip. With theuse of elimination diet, she was also found to be intolerant to a numberof other foods.

[0286] All subjects were skin prick-tested with the Hollister-Stier(Miles Inc., Elkhart, Ind.) whole cow's milk antigen.

EXAMPLE 59 Controls

[0287] Eleven control subjects were studied (9 female and 2 male). Theaverage age was 37 years. Control subjects had negative skin prick teststo Hollister-Stier whole cow's milk extract and had no history of foodintolerance, particularly to milk or cheese. Control subjects were ingood health and had no history of migraine or of functionalgastrointestinal symptoms. Controls had not ingested milk during the 3-4hour period before blood samples were taken, and were not challengedwith cow's milk.

EXAMPLE 60 Serum Samples

[0288] Blood samples were collected from milk intolerant patients andcontrols. The serum was collected and stored frozen at −70° C. in 0.5- 1ml aliquots. A fresh aliquot of serum was used for each ELISA assay.

EXAMPLE 61 Antigens

[0289] β-lactoglobulin (BL) (3 times crystallized and lyophilized),α-lactalbumin (AL) and α-s-casein (CA) were purchased from SigmaChemical Co., St. Louis. Mo. They were all made up at 2 mg/ml in PBS pH7.4 and stored frozen in aliquots at −20° C. Horse serum (HS)(Commonwealth Serum Laboratories, Melbourne, Australia) was used as anon-milk control antigen. A similar amount to the milk antigens wascoated onto ELISA plates. HS was also present in the sample and antibodydiluent.

EXAMPLE 62 Anti-TABM Antiserum

[0290] Polyclonal (rabbit) antisera (R28, R30) specific for human TABMwere prepared against TABM from human serum isolated by ammoniumsulphate precipitation and ion exchange chromatography (8). The antiserawere absorbed with fetal calf serum, immunoglobulin IgG and IgM beadsand albumin-sepharose beads to remove antibodies that could react withhuman immunoglobulins, albumin or bovine serum. There is no crossreactivity of the absorbed R28. or R30 antibody with human IgG, IgM,human or bovine albumin as tested by ELISA (8). The R28 antibody hasbeen shown to bind to TABM specific for tetanus toxoid, and T-cellproteins (immunoblot), but does not bind to T lymphocyte membranes orB-cell proteins (8).

EXAMPLE 63 Antigen Capture Enzyme Linked Immunosorbent Assay (Ag-ELISA)

[0291] Ag-ELISA was performed by a modification of that previouslydescribed (20-22). Maxisorp ELISA plates (Nunc, Denmark) were coatedwith 500 ng of milk protein in 100 μl of 0.06M carbonate buffer pH 9.6overnight at 4° C. The plates were washed 5 times with PBS (pH7.2)-0.05% v/v Tween-20 (PBS-Tw) and blocked with 200 μl per well of 4%v/v horse serum (HS) in carbonate buffer for 60 minutes at 37° C. Theplates were washed and 100 μl of the serum samples diluted in PBS-Tw-4%v/v HS (anti-IgG, {fraction (1/1,000)}: IgG1, {fraction (1/100)}; IgG2,{fraction (1/100)}: IgG3, {fraction (1/100)}: IgG4, {fraction (1/200)};IgE, {fraction (1/10)}: and TABM, ⅕) were added to the appropriate wellsand incubated for 90 minutes at 37° C. The plates were washed and 100 μlof the primary antibody [rabbit anti-human IgG (DAKO, Denmark).{fraction (1/2,500)}: mouse monoclonal anti-human IgG1, anti-IgG2,{fraction (1/1,600)}; anti-IgG3, {fraction (1/800)}: anti-IgG4,{fraction (1/3200)} (Nordic, Netherlands); rabbit anti-human IgE(Behring, Germany), {fraction (1/4,000)}; and R28 rabbit anti-human TABM{fraction (1/300)}] diluted in PBS-Tw-4% v/v HS were added to theappropriate wells and incubated for 90 minutes at 37° C. After washing,the enzyme conjugates, rabbit anti-mouse IgG peroxidase {fraction(1/400)} (DAKO, Denmark) or sheep anti-rabbit IgG peroxidase. {fraction(1/400)} (Silenus, Australia) were diluted in PBS-Tw-4% v/v HS and 100μl added to the wells. The plates were incubated at 37° C. for 90minutes. After washing, 100 μl 3,3′,5,5′-tetramethyl benzidine (TMB)substrate (KPL, Gaithersburg, Md.) was added to the wells and incubatedat room temperature. The color reaction was topped with 100 μl H₂SO₄when the optical density (O.D.) of approximately 2, for immunoglobulinlevels and 0.5 for TABM was reached. Plates were read at 450 nm. Allsamples were tested in duplicate and the mean O.D. was used for allcalculations.

EXAMPLE 64 ELISA Standards-Antibodies to Cow's Milk Proteins

[0292] A standard curve was produced using a serum pool from 50 childrenwith at least a positive RAST to milk assessed using Kallestad antigendiscs and the Kallestad Allercoat EAST kit (Sanofi Diagnostics Pasteur,Inc.). The serum pool was aliquoted and stored frozen at −70° C. Foreach assay, a fresh aliquot was thawed and serially diluted by ½. Theundiluted pool was assigned an arbitrary number of units for each of theantibody assays as follows: IgG, 10⁶; IgG1, 10⁵; IgG2, 10⁵; IgG3, 10⁵;IgG4, 10⁵; and IgE, 10⁴. The standard dilution ranges for each assaywere: IgG. {fraction (1/500)}-{fraction (1/6,400)}; IgG1, {fraction(1/100)}-{fraction (1/6,400)}; IgG2, {fraction (1/100)}-{fraction(1/6,400)}; IgGe, {fraction (1/50)}-{fraction (1/3,200)}; IgG4,{fraction (1/100)}-{fraction (1/6,400)}; and IgE, {fraction(1/10)}-{fraction (1/640)}.

EXAMPLE 65 ELISA Standards-TABM to Cow's Milk Proteins

[0293] Three separate serum pools were prepared as standards todetermine TABM levels to β-lactoglobulin, α-lactalbumin and casein. Theundiluted serum pools were assigned 100, 500, 1000 units/ml,respectively. Each of the standard serum pools consisted of an equalvolume of serum from three patients that had a high TABM response to theparticular antigen. The standards were serially diluted (½) from⅕-{fraction (1/320)}, and the appropriate number of units were assignedto correspond to the serially diluted standard. Patient serum sampleswere tested at a dilution of ⅕. This allowed for plotting a standardcurve with arbitrary units (rather than a dilution value) against theO.D. The sample O.D. values were manually read off the standard curvesas arbitrary units. Comparison of groups were performed using theMann-Whitney U Test.

EXAMPLE 66 ELISA for Tumor Necrosis Factor

[0294] A sandwich ELISA method was used to determine TNF-α levels inculture supernatants. ELISA plates (Costar, Cambridge, Mass.) werecoated overnight at 4° C. with a purified polyclonal goat anti-TNF-αcapture antibody (R&D Systems, Minn.) in 0.06 M carbonate buffer pH 9.6.The plate was washed 5 times with PBS-0.05% v/v Tween (PBS-Tw) andblocked with 200 μl of 1% w/v gelatin. Recombinant TNF-α (NIBSCHertfordshire, UK) was used as the standard and serially diluted by ½from 2000 to 0.98 pg/ml. All standards and samples were tested induplicate and incubated for 90 minutes at 37° C., followed by 5 washeswith PBS+TW. Standards and samples (tested in duplicate) were incubatedfor 90 minutes at 37° C., and the plate was washed 5 times with PBS-Tw.The mouse monoclonal second antibody (R&D Systems, Minn.) was appliedand incubated for 90 minutes at 37° C., and the plate washed 5 times. Aperoxidase-conjugated sheep anti-mouse IgG antibody (Silenus, Australia)was applied for 90 minutes at 37° C. After washing, TMB was added. Thereaction was incubated at room temperature until an O.D. ofapproximately 2 was reached and stopped with 2M H₂SO₄. Plates were readat 450 nm, and the data analyzed using the Beckman Immunofit EIA/RIAanalysis program (v 3.0).

EXAMPLE 67 ELISA Inhibition Assay (EIA) for TGF-β

[0295] Microtiter trays were coated 16 hours at room temperature with 10ng/well TGF-β1, 2 or 3 peptides (Santa Cruz Biotechnologies, Santa Cruz,Calif.). One hundred μl of 1:1000 dilution of anti-TGF-β1, 2 or 3 wasmixed with 10 μl wash buffer, 0.25-4 ng TGF-β1, 2 or 3 peptide,respectively, or 10-20 μl BL-TABM. After 1.5 hours, the trays werewashed and a 1:1000 dilution of AP-goat anti-rabbit IgG added. After 1.5hours at 37° C., the plates were washed and p-nitrophenyl phosphatesubstrate added, the optical density of each was determined after 15minutes incubation. The percent inhibition of the antibody binding tosolid phase TGF-β peptide by soluble peptide or BL-TABM was determinedby:${\% \quad {inhibition}} = {\frac{{O.D.\quad ({A405})} + {{soluble}\quad {peptide}\quad {or}\quad {BL}\text{-}{TABM}}}{{O.D.\quad ({A405})} + {10\quad \mu \quad 1\quad {wash}\quad {buffer}}} \times 100}$

[0296] A standard curve based on percent inhibition by 0.25-4 ng TGF-βpeptide was prepared; and to compute ng of TGF-β in a sample, thepercent inhibition obtained with samples was compared to that obtainedwith soluble antigenic peptides. The moles of inhibitory peptide wasthen computed as moles of TGF-β protein. Preliminary studies in thisassay have demonstrated that 1 mole of protein=2.3 moles peptide.Therefore, the final calculation is TGF-β protein =$\frac{{moles}\quad {TGF}\text{-}\beta \quad {in}\quad {sample}}{2.3}$

EXAMPLE 68 Purification of β-lactoglobulin-specific TABM (BL-TABM)

[0297] One hundred ml of blood was collected by venipuncture from afemale with a previously determined high level of antigen-specific TABMto the cow's milk protein β-lactoglobulin. The blood was allowed to clotat room temperature, centrifuged, and the serum collected and storedfrozen at −20° C. until required. Fifty-one ml of serum was precipitatedwith 43% w/v ammonium sulphate. The precipitate was collected aftercentrifuging at 9700 g for 20 minutes at 4° C. and dissolved in PBS, ½starting volume. The solution was re-precipitated at 43% w/v ammoniumsulphate, centrifuged, dissolved in PBS and dialyzed overnight againstPBS at 4° C. The sample was collected and centrifuged to remove anyprecipitate, and mixed for 2 hours at 4° C. by rotation withβ-lactoglobulin-sepharose beads (2 mg β-lactoglobulin per ml ofsepharose). The mixture was then poured onto a sintered glass funnel andwashed thoroughly with PBS. Molecules specific for β-lactoglobulin(TABM, IgG) were eluted with 0.2 M NaHCO₃, pH 9.6 and naming byinversion for 20 minutes at 4° C. The eluate was collected andimmediately neutralized (10% v/v) with 1M TRIS, pH 7.0. This sample wasdialyzed overnight at 4° C. against 100 mM TRIS-150 mM NaCl, pH 7.2.Removal of human immunoglobulin was accomplished by mixing withsepharose beads coupled with anti-human, IgG, IgA and IgM antibodies byinversion for 2 hour at 4° C. The β-lactoglobulin specific TABM wascollected as the flow-through from the sintered glass funnel. The samplewas concentrated using an Amicon stirred flow cell and YM-10 filter,n-ocytlglucoside (Sigma) was added to 30 mM. The sample was thensterilized through a 0.2μ filter and stored at 4° C.

[0298] The 9.27 fold concentrate of β-lactoglobulin specific TABM willbe referred to as: BL-TABM. A protein concentration of 435 μg/ml wasdetermined using the Biorad assay and BSA as the standard.

EXAMPLE 69 Effect of BL-TABM on TNF-α Production by Peripheral BloodMononuclear Cells (PBMNC)

[0299] Peripheral blood from a normal health (non-atopic) individual wascollected by venipuncture and anti-coagulated with preservative-freeheparin (Fisons Pty, Ltd., NSW). The blood was layered onto Ficoll(Pharmacia, Sweden) and centrifuged at 200 rpm for 20 minutes. Theplasma was removed and the PBMNC isolated and washed 3 times with PBS.The PBMNC were suspended in RPMI-1640 with 10% v/v heat inactivatedfetal calf serum (FCS), with penicillin and streptomycin (CSL,Melbourne). 2×10⁵ cells were plated in duplicate in 96-well, roundbottom tissue culture plates (NUNC, Denmark), in a final volume of 200μl following additions of medium, E. coli-LPS (DIFCO), 0-250 μg/ml:BL-TABM, 0-54.38 μg/ml and β-lactoglobulin, 0-5 μg/ml. All additive weremade up and diluted in culture medium. Controls included medium andBL-TABM diluent. Plates were incubated for 24 hours at 37° C. in a 5%v/v CO₂ incubator. Equal volumes of supernatant were collected from eachwell, pooled frozen until assayed for TNF-α by ELISA.

EXAMPLE 70 Polyacrylamide Gel Electrophoresis and Immunoblotting

[0300] Twelve μg TABM (20 μl) were mixed with 20 μl SDS-PAGE samplebuffer ±5% v/v β-2-mercaptoethanol as described (21). The mixtures wereboiled 5 minutes and 4 μl (870 ng) loaded on a 10/15% w/v PHAST(Pharmacia, Piscataway, N.J.) 10-15% w/v polyacrylamide gradient gel andelectrophoresed in a PHASE analyzer. Resolved proteins were stained bysilver stain or transferred to polyvinylpyrollidone membranes forimmunoblotting (21). The membrane was incubated for 1-5 hours at 37° C.with 5% w/v powdered milk in PBS, washed and incubated 16 hours at 4° C.with 10 ml 1:500 dilution (in ELISA wash buffer) of R30 anti-human TABMor 1:500 rabbit anti-human TGF-β-2 (Santa Cruz Biotechnologies, SantaCruz, Calif.) The membrane was then washed 4 times and incubated with 10ml of 1:1000 alkaline phosphatase goat anti-rabbit IgG. After incubationwith anti-IgG, the membrane was washed 4 times and5-bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium (NBT-BCIP)substrate was added.

EXAMPLE 71 Results

[0301] Immunoglobulin Production to Milk Proteins

[0302] Patients intolerant to milk were challenged with 400-500 ml ofcow's milk. The symptoms produced in the patient group by ingestion ofmilk are summarized in Table 8. These symptoms persisted for at least 8hours, and in some cases up to 36 hours. For each antigen, specific IgGlevels were higher in the patient group (FIG. 30) than the controlgroup. Different patients provided the highest IgG levels for theparticular antigen (see patient numbers, FIG. 30). IgG1 values weresignificantly higher for β-lactoglobulin and casein (p=0.001) in thepatient group. IgG2 levels were significantly higher for casein(p=0.048) in the patient group, but approached significant forβ-lactoglobulin. IgG3 levels were not significantly higher in thepatient group for each of the antigens. IgG4 levels were significantlyraised in the patient group to β-lactoglobulin (p=0.035), and approachedsignificance for α-lactalbumin (p=0.054). Serum samples showing hightotal IgG levels for particular antigens also showed a high level of oneor more IgG subclass to that antigen.

[0303] IgE levels specific for casein were significantly higher in thepatient group than in controls (p=0.013). Specific IgE levels were lowand there was no association between positive skin tests and specificIgE levels in the patient group.

[0304] TABM Production to Milk Proteins

[0305] Typical titration curves for TABM specific for β-lactoglobulinwere obtained for both serum and BL-TABM. A titration for TABM usingserum from a milk intolerant patient (FIG. 31) demonstrated TABM bindingto β-lactoglobulin. The titer for BL-TABM was considerably higher thanfor serum. Sera showing elevated TABM for milk proteins did not havehigh TABM for other antigens such as horse serum and benzoic acidconjugated to human serum albumin.

[0306] In the patient group, TABM levels were statistically higher foreach antigen (FIG. 32). For each particular antigen, different membersof the patient group provided the highest TABM levels. There was not aparallel rise between total IgG and TABM levels for particular sera.Also, four patients who did not show elevated IgG to β-lactoglobulin,α-lactoglobulin or casein, had high or intermediate levels of TABM toβ-lactoglobulin or casein.

[0307] Isolation of TABM Specific for β-Lactoglobulin

[0308] To isolate β-lactoglobulin-specific TABM, 51 ml of serum from apatient with a high titer of β-lactoglobulin-specific TABM were mixedwith sepharose beads conjugated with β-lactoglobulin. Approximately 2.4mg (0.063% total protein) were eluted. As shown in FIG. 33.β-lactoglobulin-specific TABM were detected in the eluate. Tocharacterise the TABM 500 ng of the protein were reduced and resolved byelectrophoresis in an SDS-polyacrylamide gradient gel. As shown in FIG.34 reduced, β-lactoglobulin-specific TABM were resolved as Mr 28,000 and46,000 polypeptides. These proteins were not observed in non-reducedproteins, but a faith Mr 110,000 band was observed.

[0309] Association of BL-TABM with TGF-β

[0310] To assay the functional activity of the affinity-purifiedBL-TABM, we determined the effects of BL-TABM on TNF-α production bynormal PBMNC. The culture of PBMNC with increasing levels of BL-TABMinduced increasing levels of TNF-α (FIG. 35). The addition of increasinglevels of β-lactoglobulin potentiated this effect (FIG. 35A). Theaddition of LPS (a strong inducer of TNF-α) resulted in even higheramounts of TNF-α produced (FIG. 35B).

[0311] Because TGF-β has been shown to induce and or promote theproduction of TNF-α, the inventors reasoned that the TNF-potentiatingactivity of BL-TABM may be due to associated TGF-β. To test thishypothesis, affinity-purified BL-TABM were used in an ELISA InibitionAssay to detect and quantitate TGF-β. As shown in FIG. 36, approximately72.8 ng/ml TGF-β1, 43.8 ng/ml TGF-β2, and 0 (<5 ng/ml) TGF-β3 weredetected by EIA. Immunoblotting of non-reduced BL-TABM with anti-TGF-β2antibodies (FIG. 37) revealed Mr 28,000 bands that are not detected byimmunoblotting with R28. Moreover, R28 does not bind recombinant humanTGF-β1 or 2 in ELISA.

[0312] In summary, the immune response to three cow's milk antigens.β-lactoglobulin (BLG). α-lactalbumin (AL) and casein (CA) was studied in15 milk-intolerant adult patients and 11 adult controls. IgG, IgE, IgGsubclasses (IgG1, IgG2, IgG3, IgG4) and T-cell derived antigen bindingmolecules (TABM) specific for each antigen were measured in both groups.In the patient group, a significant elevation of total IgG and TABMagainst each of the milk antigens was found as well as raised levels ofIgG1 to BLG and CA, IgG4 to BLG and IgE to CA. TABM specific for BLGwere isolated by affinity for BLG and found to be Mr 28,000-46,000polypeptides functionally and physically associated with TGF-β1 andTGF-β2. These results indicate a Th2-type immune response to the milkantigens in milk-intolerant individuals as compared with the controlgroup who show a pattern typical of anergy or deletion.

[0313] Those skilled in the art will appreciate that the inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described. It is to be understood that theinvention includes all such variations and modifications. The inventionalso includes all of the steps, features, compositions and compoundsreferred to or indicated in this specification, individually orcollectively, and any and all combinations of any two or more of saidsteps or features. TABLE 1 PURIFICATION OF TABM FROM COHN FRACTION IIIPROTEINS OD OD Anti- Anti- Percent TABM TABM of Protein 500 ng/ 50 ng/Total Fraction (mg) MW Range well well Protein Unfractionated 2604 — 2.0n.d. 100 Cohn fraction III (NH₄)₂SO₄ 965 — 2.0 n.d. 37 Sephacryl I80.6 >600,000 2.0 2.0 3.1 Sephacryl II 145 100,000-300,000 1.2 0.6 15Sephacryl III 322  40,000-100,000 0 0 12.3 Sephacryl IV 442  <30,000 0 016.9 Sephacryl I, 1.85 — 2.0 2.0 0.19 anti-Ig HSA absorbed

[0314] TABLE 2 Before After Toluene Challenge Toluene Challenge Prose:Immediate Recall   11 items  6 items Delayed Recall 10.5 items  6.5items Reaction Time:  212 msec 219 msec Symbol Digit:   50 items  32items Letter Cancellation: 71% correct 63% correct (6′12″) (5′40″)Stroop:* Words   73  75 Colours   94  90 Colour word  161 161

[0315] TABLE 3 Patient details and sensitivity to toluene followingcontrolled exposure. CHANGES ON CLINICAL NEUROPSYCHO- CLINICALDISABILITY REACTION TO LOGICAL (MILD, MODERATE, TOLUENE TESTING NO AGESEX OCCUPATION SEVERE) (0→+++) (0→+++) 1 45 M FACTORY WORKER MODERATE +++++ 2 38 F OFFICE WORKER MODERATE ++ ++ 3 44 F ASSSEMBLER- MILD + ++PROCESS WORKER 4 50 F WORKER AT SEVERE ++ ++ PETROLEUM DEPOT 5 36 FLAWYER MODERATE ++ ++ 6 19 F STUDENT MODERATE ++ +++ 7 18 M STUDENTMODERATE ++ ++ 8 30 M WORKER IN MODERATE ++ + PRINTING SECTION 9 49 FLABORATORY SEVERE +++ ++ ASSISTANT 10 38 M WORKED WITH SEVERE ++ ++SOLVENTS 11 44 F OFFICE WORKER MODERATE ++ ++ 12 46 F PROCESS SEVERE ++++++ WORKER 13 52 M TEACHER MODERATE ++ + 14 41 M WORKED WITH MODERATE ++++ SOLVENTS 15 27 M PRINTER MODERATE ++ ++ 16 47 M TEACHER MODERATE ++ +17 53 M TEACHER SEVERE ++ +++ 18 38 M SPRAY PAINTER MODERATE ++ ++ 19 35F HOME DUTIES MODERATE ++ +++ 20 43 F LABORATORY MILD ++ ++ ASSISTANT

[0316] TABLE 4 Results of neuropsychological testing of toluenesensitive patients. No. Pre Challenge Post Challenge Test Cases Mean(Range) Mean (Range) p Value Prose Memory 18 10.61  7.36 0.0003*(immediate) (No.   (7-14.5)  (3-12) Items recalled) Prose Memory 1812.80  8.77 0.0009* (delayed) (No. Items   (6-19.5)   (5-14.5) recalled)Reaction Time 17 207.80  238.40  0.0614 (millisecs) (158-264) (146-414)Letter Cancellation 16  4.77 4.79 NS Time (Mins) (3.48-6.12) (3.36-6.18)Letter Cancellation 16 82.31 77.56 0.0380* (% Correct) (52-99) (50-98)Digit Symbol 20 45.00 40.00 0.0099* (No. of correct items) (30-69)(25-57) Focal Length (cms) 13 28.53 29.53 NS (14-45) (14-48) STROOP(word) 16 61.81 58.93 NS (No. seconds/100  (35-190)  (32-165) words)STROOP (colour) 16 70.06 74.43 NS (No. seconds/100  (38-150)  (29-195)words) STROOP (colour- 15 120.06  119.73  NS word) (No. seconds/ (32-175)  (18-164) 100 words)

[0317] TABLE 5 Comparison of TABM levels with changes in Focal lengthand performance on STROOP (colour word) test pre and post toluenechallenge. Correlation Test p Value Coefficient† Focal Length (cms)0.0208* 0.6310 STROOP (colour word) 0.0465* 0.5209

[0318] TABLE 6 The median change in scores after exposure to toluene.Group 1 Group 2 14-25 Yrs, n = 7 Up to 13 Yrs, n = 10 Test Median(Range) Median (Range) p Value Prose Memory- 3 (−3.0-4.5) 7 (1-11)0.017* delayed, (No. items recalled). Digit Symbol Test, 1 (−7-5) 7(−7-19) 0.039* (No. of correct items). STROOP Colour 17 (3-42) −2(−31-9) 0.017* Word, (No. seconds/ 100 words).

[0319] TABLE 7 The median value of patient responses pre tolueneexposure, with respect to type of chemical exposure. Group A, n = 6Group B, n = 8 Test (Pre-exposure) Median (Range) Median (Range) p ValueProse Memory-  9.3  (7.0-11.5)  12.0  (9.5-14.5) 0.0215* Immediate, (No.items recalled). Reaction Time, 197 (183-223) 230 (190-264) 0.0441*(milliseconds).

[0320] TABLE 8 Summary of the symptoms produced after milk challenge inthe patient group. TIME OF PATIENT SYMPTOM RATING* O→+++ SYMPTOM SKINTEST STUDY NO. AGE SEX GIT RT SKIN CNS MS ONSET (MIN) (MM) 3 31 F + + 0++ 0 30 0 4 56 F 0 + 0 ++ 0 120 0 5 32 F 0 + 0 + + 30 3 9 50 F 0 0 0++ + 120 3 10 36 M ++ 0 0 + 0 30 0 11 33 F 0 + 0 ++ ++ 30 0 12 41 F 0 ++0 ++ 0 240 0 14 58 M 0 0 0 ++ 0 15 3 15 30 F + + 0 ++ 0 180 4 16 24 F +0 0 ++ 0 180 0 17 32 F 0 ++ 0 ++ 0 15 3 18 55 F 0 0 + ++ 0 180 0 19 25 F0 ++ 0 ++ + 15 0 24 51 F + + 0 +++ 0 15 0 25 41 F + + 0 ++ 0 60 0

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1. An isolated monoclonal antibody comprising a portion which is capableof specifically interacting with a T-cell antigen binding molecule(TABM), which monoclonal antibody has the same binding affinity andspecificity as monoclonal antibody MG3C9-1A12 produced by hybridoma cellline on deposit with American Type Culture Collection as Accession No.HB-12589.
 2. The monoclonal antibody of claim 1 wherein the TABM is ofanimal or avian species.
 3. The monoclonal antibody of claim 1 whereinthe TABM is of mammalian origin.
 4. The monoclonal antibody of claim 1wherein the TABM is of human origin.
 5. A hybridoma cell line producinga monoclonal antibody defined according to claim
 1. 6. A compositioncomprising a monoclonal antibody of claim 1 and one or morepharmaceutically acceptable carriers and/or diluents.
 7. The compositionof claim 6 wherein said monoclonal antibody specifically interacts witha TABM of an animal or avian species.
 8. The composition of claim 7wherein said monoclonal antibody specifically interacts with a TABM of amammalian species.
 9. The composition of claim 6 wherein said monoclonalantibody specifically interacts with a TABM of a human origin.
 10. Anantigen-binding fragment of the isolated monoclonal antibody of claim 1.11. A composition comprising the antibody-binding fragment of claim 10and one or more pharmaceutically acceptable carriers or diluents. 12.The monoclonal antibody of claim 1, wherein said monoclonal antibody islabeled with a reporter molecule capable of producing a detectablesignal.
 13. A composition comprising the monoclonal of claim 12 and oneor more pharmaceutically acceptable carriers or diluents.